Process for enzymatic hydrolysis of lignocellulosic material and fermentation of sugars

ABSTRACT

The invention relates to an integrated process for alcohol production from lignocellulosic material.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/544,827, filed 19 Jul. 2017, which is a National Stage entry ofInternational Application No. PCT/EP2016/051634, filed 27 Jan. 2016,which claims priority to European Patent Application No. 15152901.3,filed 28 Jan. 2015. Each of these applications is incorporated byreference in its entirety.

BACKGROUND Field of the Invention

The invention relates to an integrated process for alcohol productionfrom lignocellulosic material.

Description of Related Art

Lignocellulosic material is primarily composed of cellulose,hemicellulose and lignin and provides an attractive platform forgenerating alternative energy and chemical sources to fossil fuels. Thematerial is available in large amounts and can be converted into sugarswhich again can be converted into valuable fermentation products, suchas biofuel and organic acids.

Producing fermentation products from lignocellulosic material is knownin the art and generally includes the steps of pretreatment, hydrolysis,fermentation, and optionally recovery of the fermentation products.

During the hydrolysis, which may comprise the steps of liquefaction,pre-saccharification and/or saccharification, cellulose present in thelignocellulosic material is partly (typically 30 to 95%, dependable onenzyme activity and hydrolysis conditions) converted into reducingsugars by cellulolytic enzymes. The hydrolysis typically takes placeduring a process lasting 6 to 168 hours (see Kumar, S., Chem. Eng.Technol. 32 (2009), 517-526) under elevated temperatures of 45 to 70° C.and non-sterile conditions. Commonly, the sugars are then converted intovaluable fermentation products, such as ethanol and succinic acid, bymicroorganisms, like yeast.

Succinic acid is a well-known four-carbon organic acid that has highvalue, since it can be used as a precursor for many important industrialchemicals and consumer products. Currently, succinic acid is producedpetrochemically from butane through maleic anhydride. However, muchattention has recently been focused on the microbiological production ofsuccinic acid using microorganisms as an alternative to chemicalsynthesis.

In recent years, largely in response to uncertain fuel supply andefforts to reduce carbon dioxide emissions, production of ethanol fromrenewable biomass resources is becoming extremely important from theviewpoint of the global environment. Bioethanol is seen as a good fuelalternative, because the source crops can be grown renewably and in mostclimates around the world. In addition, the use of bioethanol isgenerally CO₂ neutral.

In recent years, the concept of the biorefinery has emerged. In thebiorefinery concept biomass conversion processes and technology toproduce a variety of products including fuels, power, chemicals and feedfor cattle are integrated. This way advantage of the natural differencesin the chemical and structural composition of the biomass feed stocks istaken. Careful management and utilization of materials, products andwastes are desirable, making the biorefinery concept a clear example ofindustrial symbiosis. By producing multiple products and integratingwaste treatment, biorefineries can maximize the values derived frombiomass feed stocks and turn biomass processing into real opportunities.

Optimization of processes performed within biorefineries and the overalldesign of biorefineries are crucial tools to increase efficiency ofbiorefineries and reduce their overall costs.

It is therefore desirable to include new and innovative concepts,designs and process configurations aimed at maximizing the output ofbiorefineries and reducing their overall costs.

SUMMARY

An object of the invention is to provide an improved integrated processfor alcohol production from lignocellulosic material. Optimization andimprovement lies in many features including, but not limited to,valorisation of side streams, separation of streams, (re-)use of certainmaterials and streams, conditions of enzymatic hydrolysis andfermentations, integration of a variety of conversion processes.Preferably, the integrated process for alcohol production fromlignocellulosic material comprises the steps of:

-   -   enzymatic hydrolysis of the lignocellulosic material to obtain        enzymatically hydrolysed lignocellulosic material,    -   solid/liquid separation of the enzymatically hydrolysed        lignocellulosic material to obtain at least a solid fraction and        at least a liquid fraction,    -   fermentation of the at least solid fraction and/or the at least        liquid fraction by an alcohol producing microorganism to produce        alcohol,    -   propagation of the alcohol producing microorganism by        fermentation of the at least liquid fraction and/or the at least        solid fraction,    -   propagation of an enzyme producing microorganism, and    -   production of enzymes by the enzyme producing microorganism.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

Throughout the present specification and the accompanying claims, thewords “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows. The articles “a” and “an” are used herein to referto one or to more than one (i.e. to one or at least one) of thegrammatical object of the article. By way of example, “an element” maymean one element or more than one element. The term “microorganism” asused herein means one or more microorganisms. Unless stated differently,the terms “the at least a solid fraction” and “the at least a liquidfraction” mean the at least solid fraction and the at least liquidfraction, respectively, as obtained after solid/liquid separation of theenzymatically hydrolysed lignocellulosic material. As described hereinafter a solid/liquid separation at least a solid fraction and at least aliquid fraction are obtained. “The at least solid fraction” and “the atleast liquid fraction” refer to the result of a separation step and canalso be replaced by the terms “the solid fraction” and “the liquidfraction”, respectively.

The invention relates to an integrated process of producing alcohol. Theterm “integrated process” is known to a person skilled in the art andmeans a process wherein two or more related process steps of at leasttwo separate industrial processes, which can be separately performed,are combined, so that at least one process step is common for the twoprocesses. Moreover, in an “integrated process” as defined hereinstreams, fractions and/or portions produced and/or obtained in oneindustrial process can be used in another industrial process therebyimproving the overall process efficiently more than the sum of eachindividual process. The integrated process optimizes the utilization ofbiomass and reduces by-products that otherwise would require treatment.In other words, the term “integrated process” means a combination of atleast two unit operations which exploits the interactions betweendifferent units in order to employ resources effectively, improve energyefficiency, improve material balance, maximize profit and/or minimizecosts. At least one of the two unit operations receives material and/orenergy, and may be dependent on these, from the other unit operation. Inan integrated process the interactions between different unit operationsare considered from the outset, rather than having them optimizedseparately. Process integration is not limited to the design of newplants, but it also covers retrofit design, for example new units to beinstalled in an old plant, and the operation of existing systems. Thepresent invention also provide alcohol and organic acid productionprocesses, wherein the units of such processes are completelyintegrated, and thus the processes are of low cost, simple operation andversatile due to the alternatives and interconnections within theirsteps. The integrated process is more energy and materials efficientthan the individual processes together, and, as such, it yields a higherproductivity with complete utilization and valorization of thelignocellulosic biomass.

The present invention relates to an integrated process for alcoholproduction from lignocellulosic material, wherein the process comprises:

-   -   enzymatic hydrolysis of the lignocellulosic material to obtain        enzymatically hydrolysed lignocellulosic material,    -   solid/liquid separation of the enzymatically hydrolysed        lignocellulosic material to obtain at least a solid fraction and        at least a liquid fraction,    -   fermentation of the at least solid fraction and/or the at least        liquid fraction by an alcohol producing microorganism to produce        alcohol,    -   propagation of the alcohol producing microorganism by        fermentation of the at least liquid fraction and/or the at least        solid fraction,    -   propagation of an enzyme producing microorganism, and    -   production of enzymes by the enzyme producing microorganism.

The present invention also relates to an integrated process for alcoholproduction from lignocellulosic material, wherein the process comprises:

-   -   pretreatment of the lignocellulosic material to obtain        pretreated lignocellulosic material,    -   enzymatic hydrolysis of the pretreated lignocellulosic material        to obtain enzymatically hydrolysed lignocellulosic material,    -   solid/liquid separation of the enzymatically hydrolysed        lignocellulosic material to obtain at least a solid fraction and        at least a liquid fraction,    -   fermentation of the at least solid fraction and/or the at least        liquid fraction by an alcohol producing microorganism to produce        alcohol,    -   propagation of the alcohol producing microorganism by        fermentation of the at least liquid fraction and/or the at least        solid fraction,    -   propagation of an enzyme producing microorganism, and    -   production of enzymes by the enzyme producing microorganism.

In an embodiment the integrated processes for alcohol productionaccording to the present invention may also comprise the steps offermentation of the at least liquid fraction and/or the at least solidfraction by an organic acid producing microorganism to produce anorganic acid and/or propagation of the organic acid producingmicroorganism by fermentation of the at least liquid fraction and/or theat least solid fraction. In that case the integrated process of theinvention can be seen as an integrated process for alcohol productionand organic acid production. In an embodiment the at least liquidfraction is used as substrate in the production of an organic acid bythe organic acid producing microorganism. In other words, the organicacid producing microorganism ferments the at least liquid fraction toproduce an organic acid. In an embodiment the organic acid producingmicroorganism does not ferment the at least solid fraction to produce anorganic acid. In an embodiment the alcohol produced by the alcoholproducing microorganism is used as a substrate in the fermentation bythe organic acid producing microorganism.

In an embodiment the at least liquid fraction is used as substrate inthe production of alcohol by the alcohol producing microorganism. Inother words, the alcohol producing microorganism ferments the at leastliquid fraction to produce alcohol. In an embodiment the alcoholproducing microorganism does not ferment the at least solid fraction toproduce alcohol. In an embodiment the liquid fraction obtained aftersolid/liquid separation of the lignocellulosic material and/or thepretreated lignocellulosic material is used as substrate in theproduction of alcohol by the alcohol producing microorganism. In anembodiment the at least liquid fraction and the liquid fraction obtainedafter solid/liquid separation of the lignocellulosic material and/or thepretreated lignocellulosic material is used as substrate in theproduction of alcohol by the alcohol producing microorganism. In anembodiment the enzymatically hydrolysed lignocellulosic material is usedas substrate in the production of alcohol by the alcohol producingmicroorganism. In other words, the enzymatically hydrolysedlignocellulosic material, before it is subjected to a solid/liquidseparation, is used as substrate in the production of alcohol by thealcohol producing microorganism.

In an embodiment the present invention relates to an integrated processfor alcohol production from lignocellulosic material as describedherein, wherein the process comprises the step of propagation of thealcohol producing microorganism by fermentation of the at least liquidfraction and/or the at least solid fraction. If necessary, one or moreexternal carbon and nutrient sources can be added before and/or duringthe propagation. Conditions for propagation will depend on the type ofmicroorganism used and are well within the scope of the skilled artisan.

In an embodiment the present invention relates to an integrated processfor alcohol production from lignocellulosic material as describedherein, wherein the process also comprises the step of propagation ofthe organic acid producing microorganism by fermentation of the at leastliquid fraction and/or the at least solid fraction. If necessary, one ormore external carbon and nutrient sources can be added before and/orduring the propagation. Conditions for propagation will depend on thetype of microorganism used and are well within the scope of the skilledartisan.

In an embodiment the present invention relates to an integrated processfor alcohol production from lignocellulosic material as describedherein, wherein the process comprises the step of propagation of anenzyme producing microorganism. If necessary, one or more externalcarbon and nutrient sources can be added before and/or during thepropagation. Conditions for propagation will depend on the type ofmicroorganism used and are well within the scope of the skilled artisan.

In an embodiment the present invention relates to an integrated processfor alcohol production from lignocellulosic material as describedherein, wherein the process comprises the step of production of enzymesby an enzyme producing microorganism. If necessary, one or more externalcarbon and nutrient sources can be added before and/or during theproduction. Conditions for production will depend on the type ofmicroorganism used and are well within the scope of the skilled artisan.

In an embodiment the present invention relates to an integrated processfor alcohol production from lignocellulosic material, wherein theprocess comprises:

-   -   enzymatic hydrolysis of the lignocellulosic material to obtain        enzymatically hydrolysed lignocellulosic material,    -   solid/liquid separation of the enzymatically hydrolysed        lignocellulosic material to obtain at least a solid fraction and        at least a liquid fraction,    -   fermentation of the at least solid fraction and/or the at least        liquid fraction by an alcohol producing microorganism to produce        alcohol,    -   fermentation of the at least liquid fraction and/or the at least        solid fraction by an organic acid producing microorganism to        produce an organic acid,    -   propagation of the alcohol producing microorganism by        fermentation of the at least liquid fraction and/or the at least        solid fraction,    -   propagation of the organic acid producing microorganism by        fermentation of the at least liquid fraction and/or the at least        solid fraction,    -   propagation of an enzyme producing microorganism, and    -   production of enzymes by the enzyme producing microorganism.

In an embodiment the present invention relates to an integrated processfor alcohol production from lignocellulosic material, wherein theprocess comprises:

-   -   pretreatment of the lignocellulosic material to obtain        pretreated lignocellulosic material,    -   enzymatic hydrolysis of the pretreated lignocellulosic material        to obtain enzymatically hydrolysed lignocellulosic material,    -   solid/liquid separation of the enzymatically hydrolysed        lignocellulosic material to obtain at least a solid fraction and        at least a liquid fraction,    -   fermentation of the at least solid fraction and/or the at least        liquid fraction by an alcohol producing microorganism to produce        alcohol,    -   fermentation of the at least liquid fraction and/or the at least        solid fraction by an organic acid producing microorganism to        produce an organic acid,    -   propagation of the alcohol producing microorganism by        fermentation of the at least liquid fraction and/or the at least        solid fraction,    -   propagation of the organic acid producing microorganism by        fermentation of the at least liquid fraction and/or the at least        solid fraction,    -   propagation of an enzyme producing microorganism, and    -   production of enzymes by the enzyme producing microorganism.

In an embodiment enzymatic hydrolysis and fermentation may be separatesteps, but may also be combined. Examples include, but are not limitedto, separate hydrolysis and fermentation (SHF), simultaneoussaccharification and fermentation (SSF), simultaneous saccharificationand co-fermentation (SSCF), hybrid hydrolysis and fermentation (HHF),separate hydrolysis and co-fermentation (SHCF), hybrid hydrolysis andco-fermentation (HHCF), and direct microbial conversion (DMC), alsosometimes called consolidated bioprocessing (CBP).

In an embodiment the lignocellulosic material is subjected to at leastone solid/liquid separation before the enzymatic hydrolysis. In anembodiment the pretreated lignocellulosic material is subjected to atleast one solid/liquid separation before the enzymatic hydrolysis. So,before subjecting the lignocellulosic material and/or pretreatedlignocellulosic material to enzymatic hydrolysis, it can be subjected toat least one solid/liquid separation. The methods and conditions ofsolid/liquid separation will depend on the type of lignocellulosicmaterial used and are well within the scope of the skilled artisan.Examples include, but are not limited to, centrifugation, cyclonicseparation, filtration, decantation, sieving and sedimentation. Duringsolid/liquid separation, means and/or aids for improving the separationmay be used.

In an embodiment the liquid fraction obtained after solid/liquidseparation of the lignocellulosic material and/or the pretreatedlignocellulosic material is subjected to enzymatic hydrolysis. The solidfraction obtained after solid/liquid separation of the lignocellulosicmaterial and/or the pretreated lignocellulosic material is subjected toa further solid/liquid separation. This cycle can be repeated severaltimes.

In another embodiment the solid fraction obtained after solid/liquidseparation of the lignocellulosic material and/or the pretreatedlignocellulosic material is subjected to enzymatic hydrolysis, while theliquid fraction obtained after solid/liquid separation of thelignocellulosic material and/or the pretreated lignocellulosic materialis used as substrate in at least one of the fermentation processes. Inan embodiment the liquid fraction obtained after solid/liquid separationof the lignocellulosic material and/or the pretreated lignocellulosicmaterial is used as substrate in the propagation of the alcoholproducing microorganism and/or is used as substrate in the fermentationby the alcohol producing microorganism to produce alcohol.

Before subjecting the lignocellulosic material and/or the pretreatedlignocellulosic material to a solid/liquid separation step additionalcompounds such as a centrifugation aid can be added.

In an embodiment the enzymes used in the enzymatic hydrolysis can beadded before subjecting the lignocellulosic material and/or thepretreated lignocellulosic material to a solid/liquid separation step.The enzymes then partly end up in the liquid fraction.

In an embodiment a part of the enzymatically hydrolysed lignocellulosicmaterial is used in the propagation of the enzyme producingmicroorganism and/or the production of enzymes by the enzyme producingmicroorganism. In an embodiment the part of the enzymatically hydrolysedlignocellulosic material that is used in the propagation of the enzymeproducing microorganism and/or the production of enzymes by the enzymeproducing microorganism is the at least liquid fraction obtained aftersolid/liquid separation of the enzymatically hydrolysed lignocellulosicmaterial. In an embodiment a part of the enzymatically hydrolysedlignocellulosic material and a part of the lignocellulosic materialand/or the pretreated lignocellulosic material is used in thepropagation of the enzyme producing microorganism and/or the productionof enzymes by the enzyme producing microorganism. This means that a partof the enzymatically hydrolysed lignocellulosic material and/or a partof the lignocellulosic material and/or the pretreated lignocellulosicmaterial is added to the enzyme producing microorganism before and/orduring propagation and/or before and/or during production of enzymes bythe enzyme producing microorganism. Of course, the enzyme producingmicroorganism can also be added to the part of the enzymaticallyhydrolysed lignocellulosic material and/or the part of thelignocellulosic material and/or the pretreated lignocellulosic material.The lignocellulosic material and/or the pretreated lignocellulosicmaterial used in the propagation of the enzyme producing microorganismand/or the production of enzymes by the enzyme producing microorganismhas not undergone enzymatic hydrolysis. In an embodiment the part of thelignocellulosic material and/or the pretreated lignocellulosic materialthat is used in the propagation of the enzyme producing microorganismand/or the production of enzymes by the enzyme producing microorganismhas not been subjected to a solid/liquid separation. In anotherembodiment the part of the lignocellulosic material and/or thepretreated lignocellulosic material that is used in the propagation ofthe enzyme producing microorganism and/or the production of enzymes bythe enzyme producing microorganism has been subjected to a solid/liquidseparation. In the latter case, the solid fraction obtained aftersolid/liquid separation of the lignocellulosic material and/or thepretreated lignocellulosic material is used in the propagation of theenzyme producing microorganism and/or the production of enzymes by theenzyme producing microorganism.

In a preferred embodiment the enzymes produced by the enzyme producingmicroorganism are used in the enzymatic hydrolysis of thelignocellulosic material and/or the pretreated lignocellulosic materialto obtain enzymatically hydrolysed lignocellulosic material.

In an embodiment the propagation of the enzyme producing microorganismand the production of enzymes by the enzyme producing microorganism area single step, meaning that during propagation of the enzyme producingmicroorganism enzymes are already produced by the microorganism.

The enzymatically hydrolysed lignocellulosic material that is added tothe enzyme producing microorganism before and/or during propagation ofthe enzyme producing microorganism and/or before and/or duringproduction of enzymes by the enzyme producing microorganism can beconcentrated before addition. In an embodiment the part of theenzymatically hydrolysed lignocellulosic material that is used in thepropagation of the enzyme producing microorganism and/or the productionof enzymes by the enzyme producing microorganism has been subjected to asolid/liquid separation. The liquid fraction obtained after solid/liquidseparation of the enzymatically hydrolysed lignocellulosic material maybe used in the propagation of the enzyme producing microorganism and/orthe production of enzymes by the enzyme producing microorganism. In anembodiment that liquid fraction may be subjected to a concentration stepbefore it is used in the propagation of the enzyme producingmicroorganism and/or the production of enzymes by the enzyme producingmicroorganism.

The lignocellulosic material and/or the pretreated lignocellulosicmaterial that is added to the enzyme producing microorganism beforeand/or during propagation of the enzyme producing microorganism and/orbefore and/or during production of enzymes by the enzyme producingmicroorganism can be washed before addition.

In an embodiment the ratio between the part of the enzymaticallyhydrolysed lignocellulosic material and the part of the lignocellulosicmaterial and/or the pretreated lignocellulosic material that are used inthe propagation of the enzyme producing microorganism and/or theproduction of enzymes by the enzyme producing microorganism is between1% wt: 99% wt and 99% wt: 1% wt. Of course, the ratio may differ in caseone or more external carbon sources are used in the propagation of theenzyme producing microorganism and/or the production of enzymes by theenzyme producing microorganism. In an alternative embodiment, when theenzymatic hydrolysis comprises a separate liquefaction step andsaccharification step (as described in more detail below), the productof the liquefaction step can be used in the propagation of the enzymeproducing microorganism and/or the production of enzymes by the enzymeproducing microorganism. This can be done with or without addition ofenzymatically hydrolysed lignocellulosic material. Of course, also eachand every combination of part of the enzymatically hydrolysedlignocellulosic material, part of the pretreated lignocellulosicmaterial, product of the liquefaction step and external carbon andnutrient source can be used in the propagation of the enzyme producingmicroorganism and/or the production of enzymes by the enzyme producingmicroorganism.

The part of the enzymatically hydrolysed lignocellulosic material andthe part of the lignocellulosic material and/or the pretreatedlignocellulosic material that are used in the propagation of the enzymeproducing microorganism and/or the production of enzymes by the enzymeproducing microorganism can vary. The part of the enzymaticallyhydrolysed lignocellulosic material that is used in the propagation ofthe enzyme producing microorganism and/or the production of enzymes bythe enzyme producing microorganism can be at least 1 wt %, at least 2 wt%, at least 3 wt %, at least 4 wt %, at least 5 wt %, at least 6 wt %,at least 7 wt %, at least 8 wt %, at least 9 wt %, at least 10 wt %, atleast 11 wt %, at least 12 wt %, at least 13 wt %, at least 14 wt %, atleast 15 wt %, at least 20 wt % of the total enzymatically hydrolysedlignocellulosic material.

The part of the lignocellulosic material and/or the pretreatedlignocellulosic material that is used in the propagation of the enzymeproducing microorganism and/or the production of enzymes by the enzymeproducing microorganism can be at least 1 wt %, at least 2 wt %, atleast 3 wt %, at least 4 wt %, at least 5 wt %, at least 6 wt %, atleast 7 wt %, at least 8 wt %, at least 9 wt %, at least 10 wt % of thetotal lignocellulosic material and/or the total pretreatedlignocellulosic material.

Next to the enzymatically hydrolysed lignocellulosic material and thelignocellulosic material and/or the pretreated lignocellulosic material,at least one external carbon and nutrient source can be used in thepropagation of the enzyme producing microorganism and/or the productionof enzymes by the enzyme producing microorganism. The external carbonand nutrient source can have the function of inducer and/or nutrient. Ofcourse, several different external carbon and nutrient sources may beadded. Carbon and nutrient sources suitable in the propagation of anenzyme producing microorganism and/or in the production of enzymes by anenzyme producing microorganism are known to a person skilled in the art.

After enzymatic hydrolysis, the enzymatically hydrolysed lignocellulosicmaterial is subjected to a solid/liquid separation. Methods forsolid/liquid separation include, but are not limited to, centrifugation,cyclonic separation, filtration, decantation, sieving and sedimentation.During solid/liquid separation, means and/or aids may be used to improvethe separation.

The solid/liquid separation leads to at least a solid fraction and atleast a liquid fraction. In an embodiment the at least solid fractioncomprises between 3 and 97 wt % C5 sugars. In an embodiment the at leastliquid fraction comprises between 1 and 97 wt % C6 sugars.

In an embodiment the enzymatic hydrolysis comprises at least aliquefaction step wherein the lignocellulosic material and/or thepretreated lignocellulosic material is hydrolysed in at least a firstcontainer, and a saccharification step wherein the liquefied material ishydrolysed in the at least first container and/or in at least a secondcontainer. Saccharification can be done in the same container as theliquefaction (i.e. the at least first container), it can also be done ina separate container (i.e. at least a second container). So, in theenzymatic hydrolysis of the integrated processes according to thepresent invention liquefaction and saccharification may be combined.Alternatively, the liquefaction and saccharification may be separatesteps. Liquefaction and saccharification may be performed at differenttemperatures, but may also be performed at a single temperature. In anembodiment the temperature of the liquefaction is higher than thetemperature of the saccharification. Liquefaction is preferably carriedout at a temperature of 60-75° C. and saccharification is preferablycarried out at a temperature of 50-65° C.

The enzymatic hydrolysis can be performed in one or more containers, butcan also be performed in one or more tubes or any other continuoussystem. This also holds true when the enzymatic hydrolysis comprises aliquefaction step and a saccharification step. The liquefaction step canbe performed in one or more containers, but can also be performed in oneor more tubes or any other continuous system and/or the saccharificationstep can be performed in one or more containers, but can also beperformed in one or more tubes or any other continuous system. Examplesof containers to be used in the present invention include, but are notlimited to, fed-batch stirred containers, batch stirred containers,continuous flow stirred containers with ultrafiltration, and continuousplug-flow column reactors. Stirring can be done by one or moreimpellers, pumps and/or static mixers.

In an embodiment the lignocellulosic material and/or the pretreatedlignocellulosic material can be added to the one or more containers usedfor the enzymatic hydrolysis. In an embodiment the enzymes used in theenzymatic hydrolysis are already present in the one or more containersbefore the lignocellulosic material and/or the pretreatedlignocellulosic material is added. In another embodiment the enzymesused in the enzymatic hydrolysis can be added to the one or morecontainers. In an embodiment the lignocellulosic material and/or thepretreated lignocellulosic material is already present in the one ormore containers before the enzymes used in the enzymatic hydrolysis areadded. In an embodiment both the lignocellulosic material and/or thepretreated lignocellulosic material and the enzymes used in theenzymatic hydrolysis are added simultaneously to the one or morecontainers. The enzymes used in the enzymatic hydrolysis may be anaqueous composition. This paragraph also holds true when the enzymatichydrolysis comprises a liquefaction step and a saccharification step.

The enzymes used in the enzymatic hydrolysis may be added before and/orduring the enzymatic hydrolysis. As indicated above, when thelignocellulosic material and/or the pretreated lignocellulosic materialis subjected to a solid/liquid separation before enzymatic hydrolysis,the enzymes used in the enzymatic hydrolysis may be added before thesolid/liquid separation. Alternatively, they may also be added aftersolid/liquid separation or before and after solid/liquid separation. Theenzymes may also be added during the enzymatic hydrolysis. In case theenzymatic hydrolysis comprises a liquefaction step and saccharificationstep, additional enzymes may be added during and/or after theliquefaction step. The additional enzymes may be added before and/orduring the saccharification step. Additional enzymes may also be addedafter the saccharification step.

In an embodiment the total enzymatic hydrolysis time is 10 hours ormore, 12 hours or more, 14 hours or more, 16 hours or more, 18 hours ormore, 20 hours or more, 30 hours or more, 40 hours or more, 50 hours ormore, 60 hours or more, 70 hours or more, 80 hours or more, 90 hours ormore, 100 hours or more, 110 hours or more, 120 hours or more, 130 hoursor more, 140 hours or more, 150 hours or more, 160 hours or more, 170hours or more, 180 hours or more, 190 hours or more, 200 hours or more.

In an embodiment, the total enzymatic hydrolysis time is 10 to 300hours, 16 to 275 hours, preferably 20 to 250 hours, more preferably 30to 200 hours, most preferably 40 to 150 hours.

The viscosity of the lignocellulosic material in the one or morecontainers used for the enzymatic hydrolysis is kept between 10 and 4000cP, between 10 and 2000 cP, preferably between 10 and 1000 cP.

In case the integrated process comprises an enzymatic hydrolysiscomprising a liquefaction step and a saccharification step, theviscosity of the lignocellulosic material in the liquefaction step iskept between 10 and 4000 cP, between 10 and 2000 cP, preferably between10 and 1000 cP and/or the viscosity of the lignocellulosic material inthe saccharification step is kept between 10 and 1000 cP, between 10 and900 cP, preferably between 10 and 800 cP.

The viscosity can be determined with a Brookfield DV III Rheometer atthe temperature used for the hydrolysis.

In an embodiment oxygen is added during the enzymatic hydrolysis. In anembodiment oxygen is added during at least a part of the enzymatichydrolysis. Oxygen can be added continuously or discontinuously duringthe enzymatic hydrolysis. In an embodiment oxygen is added one or moretimes during the enzymatic hydrolysis. In an embodiment oxygen may beadded before the enzymatic hydrolysis, during the addition oflignocellulosic material to a container used of enzymatic hydrolysis,during the addition of enzyme to a container used of enzymatichydrolysis, during a part of the enzymatic hydrolysis, during the wholeenzymatic hydrolysis or any combination thereof. Oxygen is added to theone or more containers used in the enzymatic hydrolysis.

Oxygen can be added in several forms. For example, oxygen can be addedas oxygen gas, oxygen-enriched gas, such as oxygen-enriched air, or air.Oxygen may also be added by means of in situ oxygen generation. Forexample, oxygen may be generated by electrolysis, oxygen may be producedenzymatically, e.g. by the addition of peroxide, or oxygen may beproduced chemically, e.g. by an oxygen generating system such as KHSO₅.For example, oxygen is produced from peroxide by catalase. The peroxidecan be added in the form of dissolved peroxide or generated by anenzymatic or chemical reaction. In case catalase is used as enzyme toproduce oxygen, catalase present in the enzyme composition for thehydrolysis can be used or catalase can be added for this purpose.

Examples how to add oxygen include, but are not limited to, addition ofoxygen by means of sparging, electrolysis, chemical addition of oxygen,filling the one or more containers used in the enzymatic hydrolysis fromthe top (plunging the hydrolysate into the tank and consequentlyintroducing oxygen into the hydrolysate) and addition of oxygen to theheadspace of said one or more containers. When oxygen is added to theheadspace of the container(s), sufficient oxygen necessary for thehydrolysis reaction may be supplied. In general, the amount of oxygenadded to the container(s) can be controlled and/or varied. Restrictionof the oxygen supplied is possible by adding only oxygen during part ofthe hydrolysis time in said container(s). Another option is addingoxygen at a low concentration, for example by using an mixture of airand recycled air (air leaving the container) or by “diluting” air withan inert gas. Increasing the amount of oxygen added can be achieved byaddition of oxygen during longer periods of the hydrolysis time, byadding the oxygen at a higher concentration or by adding more air.Another way to control the oxygen concentration is to add an oxygenconsumer and/or an oxygen generator. Oxygen can be introduced, forexample blown, into the liquid hydrolysis container contents oflignocellulosic material. It can also be blown into the headspace of thecontainer.

In an embodiment oxygen is added to the one or more containers used inthe enzymatic hydrolysis before and/or during and/or after the additionof the lignocellulosic material and/or the pretreated lignocellulosicmaterial to said one or more containers. The oxygen may be introducedtogether with the lignocellulosic material and/or the pretreatedlignocellulosic material that enters the hydrolysis container(s). Theoxygen may be introduced into the material stream that will enter thecontainer(s) or with part of the container(s) contents that passes anexternal loop of the container(s).

In the enzymatic hydrolysis amorphous and crystalline polysaccharides orcellulose are hydrolysed to sugars such as glucose. Amorphouspolysaccharides are for example converted to oligosaccharides byendoglucanases and then the oligosaccharides can be converted bycellobiohydrolases and beta-glucosidases to glucose. The conversion ofthe crystalline polysaccharides may occur in parallel or sequential andcontinue even when most of the amorphous polysaccharides are hydrolysed.The addition of oxygen in combination with lytic polysaccharidemonooxygenases is beneficial during the hydrolysis of the crystallinepolysaccharides for example in the degradation of the polysaccharidesinto oligosaccharides. The crystalline glucan structure can be opened bylytic polysaccharide monooxygenases. This type of enzyme opens up thestructure by oxidizing the glycosidic bonds and making it accessible forthe other cellulolytic enzymes for further hydrolysing theoligosaccharides into glucose. The addition of oxygen is very useful,especially in the phase wherein crystalline polysaccharides areconverted by enzymes.

In an embodiment the container(s) used in the enzymatic hydrolysis ofthe integrated processes of the present invention have a volume of atleast 1 m³. Preferably, the containers have a volume of at least 1 m³,at least 2 m³, at least 3 m³, at least 4 m³, at least 5 m³, at least 6m³, at least 7 m³, at least 8 m³, at least 9 m³, at least 10 m³, atleast 15 m³, at least 20 m³, at least 25 m³, at least 30 m³, at least 35m³, at least 40 m³, at least 45 m³, at least 50 m³, at least 60 m³, atleast 70 m³, at least 75 m³, at least 80 m³, at least 90 m³, at least100 m³, at least 200 m³, at least 300 m³, at least 400 m³, at least 500m³, at least 600 m³, at least 700 m³, at least 800 m³, at least 900 m³,at least 1000 m³, at least 1500 m³, at least 2000 m³, at least 2500 m³.In general, the container(s) will be smaller than 3000 m³ or 5000 m³. Incase several containers are used in the enzymatic hydrolysis of theintegrated processes of the present invention, they may have the samevolume, but also may have a different volume. In case the enzymatichydrolysis of the integrated processes of the present inventioncomprises a separate liquefaction step and saccharification step thecontainer(s) used for the liquefaction step and the container(s) usedfor the saccharification step may have the same volume, but also mayhave a different volume.

In an embodiment the container(s) used in the fermentation of the atleast solid fraction and/or the at least liquid fraction by an alcoholproducing microorganism to produce alcohol have a volume of at least 1m³. Preferably, the containers have a volume of at least 1 m³, at least2 m³, at least 3 m³, at least 4 m³, at least 5 m³, at least 6 m³, atleast 7 m³, at least 8 m³, at least 9 m³, at least 10 m³, at least 15m³, at least 20 m³, at least 25 m³, at least 30 m³, at least 35 m³, atleast 40 m³, at least 45 m³, at least 50 m³, at least 60 m³, at least 70m³, at least 75 m³, at least 80 m³, at least 90 m³, at least 100 m³, atleast 200 m³, at least 300 m³, at least 400 m³, at least 500 m³, atleast 600 m³, at least 700 m³, at least 800 m³, at least 900 m³, atleast 1000 m³, at least 1500 m³, at least 2000 m³, at least 2500 m³, atleast 3000 m³, at least 3500 m³, at least 4000 m³, at least 4500 m³. Ingeneral, the container(s) will be smaller than 5000 m³.

In an embodiment the container(s) used in the fermentation of the atleast liquid fraction and/or the at least solid fraction by an organicacid producing microorganism to produce an organic acid have a volume ofat least 1 m³. Preferably, the containers have a volume of at least 1m³, at least 2 m³, at least 3 m³, at least 4 m³, at least 5 m³, at least6 m³, at least 7 m³, at least 8 m³, at least 9 m³, at least 10 m³, atleast 15 m³, at least 20 m³, at least 25 m³, at least 30 m³, at least 35m³, at least 40 m³, at least 45 m³, at least 50 m³, at least 60 m³, atleast 70 m³, at least 75 m³, at least 80 m³, at least 90 m³, at least100 m³, at least 200 m³, at least 300 m³, at least 400 m³, at least 500m³, at least 600 m³, at least 700 m³, at least 800 m³, at least 900 m³,at least 1000 m³, at least 1500 m³. In general, the container(s) will besmaller than 2000 m³.

In an embodiment the container(s) used in the propagation of the alcoholproducing microorganism by fermentation of the at least liquid fractionand/or the at least solid fraction have a volume of at least 1 m³.Preferably, the containers have a volume of at least 1 m³, at least 2m³, at least 3 m³, at least 4 m³, at least 5 m³, at least 6 m³, at least7 m³, at least 8 m³, at least 9 m³, at least 10 m³, at least 15 m³, atleast 20 m³, at least 25 m³, at least 30 m³, at least 35 m³, at least 40m³, at least 45 m³, at least 50 m³, at least 60 m³, at least 70 m³, atleast 75 m³, at least 80 m³, at least 90 m³, at least 100 m³, at least200 m³, at least 300 m³, at least 400 m³. In general, the container(s)will be smaller than 500 m³.

In an embodiment the container(s) used in the propagation of the organicacid producing microorganism by fermentation of the at least liquidfraction and/or the at least solid fraction have a volume of at least 1m³. Preferably, the containers have a volume of at least 1 m³, at least2 m³, at least 3 m³, at least 4 m³, at least 5 m³, at least 6 m³, atleast 7 m³, at least 8 m³, at least 9 m³, at least 10 m³, at least 15m³, at least 20 m³, at least 25 m³, at least 30 m³, at least 35 m³, atleast 40 m³, at least 45 m³, at least 50 m³, at least 60 m³, at least 70m³, at least 75 m³, at least 80 m³, at least 90 m³, at least 100 m³, atleast 150 m³. In general, the container(s) will be smaller than 200 m³.

In an embodiment the container(s) used in the propagation of an enzymeproducing microorganism have a volume of at least 1 m³. Preferably, thecontainers have a volume of at least 1 m³, at least 2 m³, at least 3 m³,at least 4 m³, at least 5 m³, at least 6 m³, at least 7 m³, at least 8m³, at least 9 m³, at least 10 m³, at least 15 m³, at least 20 m³, atleast 25 m³, at least 30 m³, at least 35 m³, at least 40 m³, at least 45m³, at least 50 m³, at least 60 m³, at least 70 m³, at least 75 m³, atleast 80 m³, at least 90 m³, at least 100 m³, at least 200 m³, at least300 m³, at least 400 m³. In general, the container(s) will be smallerthan 500 m³.

In an embodiment the container(s) used in the production of enzymes bythe enzyme producing microorganism have a volume of at least 1 m³.Preferably, the containers have a volume of at least 1 m³, at least 2m³, at least 3 m³, at least 4 m³, at least 5 m³, at least 6 m³, at least7 m³, at least 8 m³, at least 9 m³, at least 10 m³, at least 15 m³, atleast 20 m³, at least 25 m³, at least 30 m³, at least 35 m³, at least 40m³, at least 45 m³, at least 50 m³, at least 60 m³, at least 70 m³, atleast 75 m³, at least 80 m³, at least 90 m³. In general, thecontainer(s) will be smaller than 100 m³.

In an embodiment the enzyme producing microorganism is a fungus. In anembodiment the enzymes are derived from a filamentous fungus or theenzymes comprise a filamentous fungal enzyme. In a preferred embodimentthe fungus is Rasamsonia, with Rasamsonia emersonii being mostpreferred. The enzymes used in the enzymatic hydrolysis of theintegrated processes of the present invention are derived from a fungusor the enzymes used in the enzymatic hydrolysis of the integratedprocesses of the present invention comprise a fungal enzyme.“Filamentous fungi” include all filamentous forms of the subdivisionEumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworthand Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK). The filamentous fungiare characterized by a mycelial wall composed of chitin, cellulose,glucan, chitosan, mannan, and other complex polysaccharides. Vegetativegrowth is by hyphal elongation and carbon catabolism is obligatoryaerobic. Filamentous fungal strains include, but are not limited to,strains of Acremonium, Agaricus, Aspergillus, Aureobasidium, Beauvaria,Cephalosporium, Ceriporiopsis, Chaetomium paecilomyces, Chrysosporium,Claviceps, Cochiobolus, Coprinus, Cryptococcus, Cyathus, Emericella,Endothia, Endothia mucor, Filibasidium, Fusarium, Geosmithia,Gilocladium, Humicola, Magnaporthe, Mucor, Myceliophthora, Myrothecium,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces,Panerochaete, Pleurotus, Podospora, Pyricularia, Rasamsonia, Rhizomucor,Rhizopus, Scylatidium, Schizophyllum, Stagonospora, Talaromyces,Thermoascus, Thermomyces, Thielavia, Tolypocladium, Trametes pleurotus,Trichoderma and Trichophyton.

Several strains of filamentous fungi are readily accessible to thepublic in a number of culture collections, such as the American TypeCulture Collection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL). Examples of such strains includeAspergillus niger CBS 513.88, Aspergillus oryzae ATCC 20423, IFO 4177,ATCC 1011, ATCC 9576, ATCC14488-14491, ATCC 11601, ATCC12892, P.chrysogenum CBS 455.95, Penicillium citrinum ATCC 38065, Penicilliumchrysogenum P2, Talaromyces emersonii CBS 393.64, Acremonium chrysogenumATCC 36225 or ATCC 48272, Trichoderma reesei ATCC 26921 or ATCC 56765 orATCC 26921, Aspergillus sojae ATCC11906, Chrysosporium lucknowense C1,Garg 27K, VKM F-3500-D, ATCC44006 and derivatives thereof.

The enzymatic hydrolysis of the integrated processes of the presentinvention are advantageously applied in combination with enzymes derivedfrom a microorganism of the genus Rasamsonia or the enzymes used in theenzymatic hydrolysis of the integrated processes of the presentinvention comprise a Rasamsonia enzyme.

The enzymatic hydrolysis of the first stage is preferably done at 50−90°C. In this step thermostable cellulolytic enzymes are preferred. A“thermostable” enzyme as used herein means that the enzyme has atemperature optimum of 50° C. or higher, 60° C. or higher, 70° C. orhigher, 75° C. or higher, 80° C. or higher, 85° C. or higher. They mayfor example be isolated from thermophilic microorganisms or may bedesigned by the skilled person and artificially synthesized. In oneembodiment the polynucleotides may be isolated or obtained fromthermophilic or thermotolerant filamentous fungi or isolated fromnon-thermophilic or non-thermotolerant fungi, but are found to bethermostable.

By “thermophilic fungus” is meant a fungus that grows at a temperatureof 50° C. or higher. By “themotolerant” fungus is meant a fungus thatgrows at a temperature of 45° C. or higher, having a maximum near 50° C.

Suitable thermophilic or thermotolerant fungal cells may be a Humicola,Rhizomucor, Myceliophthora, Rasamsonia, Talaromyces, Thermomyces,Thermoascus or Thielavia cell, preferably a Rasamsonia cell. Preferredthermophilic or thermotolerant fungi are Humicola grisea var.thermoidea, Humicola lanuginosa, Myceliophthora thermophila, Papulasporathermophilia, Rasamsonia byssochlamydoides, Rasamsonia emersonii,Rasamsonia argillacea, Rasamsonia eburnean, Rasamsonia brevistipitata,Rasamsonia cylindrospora, Rhizomucor pusillus, Rhizomucor miehei,Talaromyces bacillisporus, Talaromyces leycettanus, Talaromycesthermophilus, Thermomyces lenuginosus, Thermoascus crustaceus,Thermoascus thermophilus Thermoascus aurantiacus and Thielaviaterrestris.

Thermophilic fungi are not restricted to a specific taxonomic order andoccur all over the fungal tree of life. Examples are Rhizomucor in theMucorales, Myceliophthora in Sordariales and Talaromyces, Thermomycesand Thermoascus in the Eurotiales (see Mouchacca, 1997). The majority ofTalaromyces species are mesophiles, but exceptions are species withinsections Emersonii and Thermophila. Section Emersonii includesTalaromyces emersonii, Talaromyces byssochlamydoides, Talaromycesbacillisporus and Talaromyces leycettanus, all of which grow well at 40°C. Talaromyces bacillisporus is thermotolerant, Talaromyces leycettanusis thermotolerant to thermophilic, and Talaromyces emersonii andTalaromyces byssochlamydoides are truly thermophilic (see Stolk andSamson, 1972). The sole member of Talaromyces section Thermophila,Talaromyces thermophilus, grows rapidly at 50° C. (see Stolk and Samson,1972). The current classification of these thermophilic Talaromycesspecies is mainly based on phenotypic and physiological characters, suchas their ability to grow above 40° C., ascospore color, the structure ofascornatal covering and the formation of a certain type of anamorph.Stolk and Samson (1972) stated that the members of the section Emersoniihave anamorphs of either Paecilomyces (Talaromyces byssochlamydoides andTalaromycesleycettanus) or Penicillium cylindrosporum series(Talaromyces emersonii and Talaromyces bacillisporus). Later, Pitt(1979) transferred the species belonging to the Penicilliumcylindrosporum series to the genus Geosmithia, based on variouscharacters such as the formation of conidia from terminal pores insteadof on collula (necks), a character of Penicillium and Paecilomyces.Within the genus Geosmithia, only Geosmithia argillacea isthermotolerant, and Stolk et al. (1969) and Evans (1971) proposed aconnection with members of Talaromyces sect. Emersonii. The phylogeneticrelationship of the themophilic Talaromyces species within Talaromycesand the Trichocomaceae is unknown. (see J. Houbraken, Antonie vanLeeuwenhoek 2012 February; 101(2): 403-21).

Rasamsonia is a new genus comprising thermotolerant and thermophilicTalaromyces and Geosmithia species (J. Houbraken et al., vida supra).Based on phenotypic, physiological and molecular data, Houbraken et al.proposed to transfer the species Talaromyces emersonii, Talaromycesbyssochiamydoides, Talaromyces eburneus, Geosmithia argillacea andGeosmithia cylindrospora to Rasamsonia gen. nov.

Preferred thermophilic fungi are Rasamsonia byssochiamydoides,Rasamsonia emersonii, Thermomyces lenuginosus, Talaromyces thermophilus,Thermoascus crustaceus, Thermoascus thermophilus and Thermoascusaurantiacus, with Rasamsonia emersonii being most preferred. Talaromycesemersonii, Penicillium geosmithia emersonii and Rasamsonia emersonii areused interchangeably herein.

Cellulolytic enzymes of Rasamsonia applied on pretreated lignocellulosicfeedstock show maximal conversion rates at temperature within the rangeof 50 to 70° C. The enzymes remain active under these circumstances for14 days and more without complete cessation of activity. By usingoptimal temperature conditions, a maximal amount of reducing sugars canbe released from lignocellulosic material (total hydrolysis) within theshortest possible hydrolysis time. In this way, 100% conversion ofcellulose in glucose can be achieved in less than 5 days. Thetheoretical maximum yield (Yps max in g product per gram glucose) of afermentation product can be derived from textbook biochemistry. Forethanol, 1 mole of glucose (180 g) yields according to normal glycolysisfermentation pathway in yeast 2 moles of ethanol (=2×46=92 g ethanol).The theoretical maximum yield of ethanol on glucose is therefore92/180=0.511 g ethanol/g glucose. For butanol (MW 74 g/mole) orisobutanol, the theoretical maximum yield is 1 mole of butanol per moleof glucose. So Yps max for (iso-)butanol=74/180=0.411 g (iso-)butanol/gglucose. For lactic acid the fermentation yield for homolacticfermentation is 2 moles of lactic acid (MW=90 g/mole) per mole ofglucose. According to this stoichiometry, the Yps max=1 g lactic acid/gglucose. The theoretical maximum yield of succinic acid on glucose is1.12 g succinic acid/g glucose. For other fermentation products asimilar calculation may be made. The cost reduction achieved withapplying cellulolytic enzymes of Rasamsonia are the result of an overallprocess time reduction.

Due to the high stability of the enzymes used in the processes of thepresent invention, it is possible to lower the enzyme dosage and extendthe use of the enzyme by prolonging the hydrolysis times. For example,0.175 mL enzyme/g lignocellulosic material dry matter results in releaseof approximately 90% of the theoretical maximum of reducing sugars frompretreated lignocellulosic material within 72 h. When using 0.075 mLenzyme/g lignocellulosic material dry matter, approximately 90%conversion of the theoretical maximum is achieved within 120 h. Theresults show that, because of the stability of the enzyme activity,lowering the enzyme dosage can be compensated by extending thehydrolysis time to obtain the same amount of reducing sugars. The costreduction achieved by using stable cellulolytic enzymes, such as thoseof Rasamsonia, results in lower enzyme dosages that nevertheless resultin similar hydrolysis conversion yields.

In a common process for converting lignocellulosic material intoethanol, process steps are preferably done under septic conditions tolower the operational costs. Contamination and growth of contaminatingmicroorganisms can therefore occur and result in undesirable sideeffects, such as lactic acid, formic acid and acetic acid production,yield losses of ethanol on substrate, production of toxins andextracellular polysaccharides. These effects may affect production costssignificantly. A high process temperature and/or a short process timelimits the risk on contamination during hydrolysis and fermentation.Thermostable enzymes, like those of Rasamsonia, are capable ofhydrolysing lignocellulosic material at temperatures of higher than 60°C. At these temperatures, the risk that a contaminating microorganismwill cause undesired side effects is little to almost zero.

During the fermentation step, in which ethanol is produced, temperaturesare typically between 30 to 38° C. and are preferably not raised becauseof production losses. By applying short fermentation process times, therisks and effects of contamination and/or growth of contaminants arereduced as much as possible. With stable enzymes, like those ofRasamsonia, a short fermentation time can be applied and thus risks ofcontamination and/or growth of contaminants are reduced as much aspossible. The cost reduction achieved with applying thermostablecellulolytic enzymes of Rasamsonia in this way, results in a lower riskof process failures due to contamination.

The first step after thermal pretreatment is to cool the pretreatedmaterial to temperatures wherein the enzymes have an optimal activity.On large scale, this is typically done by adding (cooled) water, which,besides decreasing the temperature, reduces the dry matter content. Byusing thermostable enzymes, like those of Rasamsonia, cost reduction canbe achieved, because (i) less cooling of the pretreated material isrequired since higher temperatures are allowed during hydrolysis, and(ii) less water is added, which increases the dry matter content duringhydrolysis and fermentation and thus increase the ethanol productioncapacity (amount produced per time unit per volume) of an ethanol plant.By using thermostable enzymes, like those of Rasamsonia, cost reductionmay also be achieved by using cooling water having a higher temperaturethan the water that is used in a process with non-thermostable enzyme.

At the end of the hydrolysis, enzyme activities appear to be low, sincelittle reducing sugars are released once almost all cellulose isconverted. The amount of enzymatic activity present, however, hasdecreased only a little, assumingly mainly due to absorption of theenzymes to the substrate. By applying solid-liquid separation afterhydrolysis, such as centrifugation, filtration, cantation,sedimentation, 60% or more (e.g. 70%) of the enzyme activity in solutioncan be recovered and re-used for hydrolysis of a new pretreatedlignocellulosic material during the next hydrolysis. Moreover, aftersolid-liquid separation the enzyme in solution can be separated from thesolution containing reducing sugars and other hydrolysis products fromthe enzymatic actions. This separation can be done by techniquesincluding, but not limited to, ultra- and microfiltration,centrifugation, cantation, sedimentation, with or without firstadsorption of the enzyme to a carrier of any kind. For example, afterhydrolysis of pretreated material with 0.175 mL/g material dry matterenzyme load for 20 h, 50% of the theoretical maximum amount of reducingsugars is liberated and after the same hydrolysis for 72 h, 90% of thetheoretical maximum amount of reducing sugars is liberated. Bycentrifugation and ultrafiltration, 60-70% of the enzyme activity wasrecovered in the retentate, while the filtrate contained more than 80%of the liberated reducing sugars. By re-using the retentate, either asit is or after further purification and/or concentration, enzyme dosageduring the next hydrolysis step can be reduced with 60 to 70%. The costreduction achieved by using stable cellulolytic enzymes, such as thoseof Rasamsonia, in this way is the consequence of a lower enzyme dosage.

The integrated processes of the present invention can be combined withenzyme recycling after hydrolysis, recycling of the ethanol producingmicroorganism after fermentation and/or recycling of the organic acidproducing microorganism after fermentation and/or recycling of theenzyme producing microorganism after production of the enzymes.

The thermostability of enzymes, like those from Rasamsonia, causesremaining cellulolytic activity after hydrolysis, fermentation andvacuum distillation in the thin stillage. The total activity of theenzyme is reduced during the three successive process steps. The thinstillage obtained after vacuum distillation can thus be re-used as asource of enzyme for a newly startedhydrolysis-fermentation-distillation process cycle of pretreatedmaterial conversion into ethanol. The thin stillage can be used eitherin concentrated or (un)diluted form and/or purified and with or withoutadditional enzyme supplementation.

In an optimal process, an amount of enzyme is supplemented into the thinstillage, before its re-use in a new process cycle, equal to the amountof activity lost during the three successive process steps of theprevious process cycle. In this way over dosage of enzyme is avoided andthus most efficient use of enzyme is obtained. Moreover, by providinghigh enzyme dosage in the first process cycle, and supplementing enzymeequal to the amount of activity lost during the three successive processsteps in the following process cycles, highest possible hydrolysis ratescan be obtained in each process cycle resulting in short hydrolysistimes of less than 48 h in combination with most efficient use ofenzymes.

By applying mixing during hydrolysis, enzymes come more often in contactwith substrates, which results in a more efficient use of the catalyticactivity. This will result in a lower enzyme dosages and thus in lowercosts, unless the mixing has a negative effect on the enzymes. Stableenzymes, like the thermostable enzymes from Rasamsonia, are robust andcan resist circumstances of (locally) high shear and temperatures, whichis the case during intensive mixing of slurries. The use of them inmixed systems is therefore beneficial and will lead to dosage and thuscosts reduction.

An advantage of expression and production of the enzymes (for example atleast two, three or four different cellulases) in a suitablemicroorganism may be a high enzyme composition yield which can be usedin the processes of the present invention.

In the processes of the present invention enzyme compositions are used.Preferably, the compositions are stable. “Stable enzyme compositions” asused herein means that the enzyme compositions retain activity after 30hours of hydrolysis reaction time, preferably at least 10%, 20%, 30%,40%, 50%, 60%, 65%, 70%, 75%, 80% 85%, 90%, 95%, 96%, 97%, 98%, 99% or100% of its initial activity after 30 hours of hydrolysis reaction time.Preferably, the enzyme composition retains activity after 40, 50, 60,70, 80, 90 100, 150, 200, 250, 300, 350, 400, 450, 500 hours ofhydrolysis reaction time.

The enzymes may be prepared by fermentation of a suitable substrate witha suitable microorganism, e.g. Rasamsonia emersonii or Aspergillusniger, wherein the enzymes are produced by the microorganism. Themicroorganism may be altered to improve or to make the enzymes. Forexample, the microorganism may be mutated by classical strainimprovement procedures or by recombinant DNA techniques. Therefore, themicroorganisms mentioned herein can be used as such to produce theenzymes or may be altered to increase the production or to producealtered enzymes which might include heterologous enzymes, e.g.cellulases, thus enzymes that are not originally produced by thatmicroorganism. Preferably, a fungus, more preferably a filamentousfungus is used to produce the enzymes. Advantageously, a thermophilic orthermotolerant microorganism is used. Optionally, a substrate is usedthat induces the expression of the enzymes by the enzyme producingmicroorganism.

The enzymes are used to release sugars from lignocellulosic material,that comprises polysaccharides. The major polysaccharides are cellulose(glucans), hemicelluloses (xylans, heteroxylans and xyloglucans). Inaddition, some hemicellulose may be present as glucomannans, for examplein wood-derived lignocellulosic material. The enzymatic hydrolysis ofthese polysaccharides to soluble sugars, including both monomers andmultimers, for example glucose, cellobiose, xylose, arabinose,galactose, fructose, mannose, rhamnose, ribose, galacturonic acid,glucoronic acid and other hexoses and pentoses occurs under the actionof different enzymes acting in concert. By sugar product is meant theenzymatic hydrolysis product of the lignocellulosic material. The sugarproduct comprises soluble sugars, including both monomers and multimers.Preferably, it comprises glucose. Examples of other sugars arecellobiose, xylose, arabinose, galactose, fructose, mannose, rhamnose,ribose, galacturonic acid, glucoronic acid and other hexoses andpentoses. The sugar product may be used as such or may be furtherprocessed, for example recovered, concentrated and/or purified.

In addition, pectins and other pectic substances such as arabinans maymake up considerably proportion of the dry mass of typically cell wallsfrom non-woody plant tissues (about a quarter to half of dry mass may bepectins).

Cellulose is a linear polysaccharide composed of glucose residues linkedby β-1,4 bonds. The linear nature of the cellulose fibers, as well asthe stoichiometry of the β-linked glucose (relative to a) generatesstructures more prone to inter strand hydrogen bonding than the highlybranched α-linked structures of starch. Thus, cellulose polymers aregenerally less soluble and form more tightly bound fibers than thefibers found in starch.

Enzymes that may be used in the invention are described in more detailbelow.

Lytic polysaccharide monooxygenases, endoglucanases (EG) andexo-cellobiohydrolases (CBH) catalyze the hydrolysis of insolublecellulose to products such as cellooligosaccharides (cellobiose as amain product), while β-glucosidases (BG) convert the oligosaccharides,mainly cellobiose and cellotriose, to glucose.

Hemicellulose is a complex polymer, and its composition often varieswidely from organism to organism and from one tissue type to another. Ingeneral, a main component of hemicellulose is β-1,4-linked xylose, afive carbon sugar. However, this xylose is often branched at 0 to 3and/or 0 to 2 atoms of xylose, and can be substituted with linkages toarabinose, galactose, mannose, glucuronic acid, galacturonic acid or byesterification to acetic acid (and esterification of ferulic acid toarabinose). Hemicellulose can also contain glucan, which is a generalterm for β-linked six carbon sugars (such as the β-(1,3)(1,4) glucansand heteroglucans mentioned previously) and additionally glucomannans(in which both glucose and mannose are present in the linear backbone,linked to each other by β-linkages).

Xylanases together with other accessory enzymes, for exampleα-L-arabinofuranosidases, feruloyl and acetylxylan esterases,glucuronidases, and β-xylosidases) catalyze the hydrolysis ofhemicellulose.

Pectic substances include pectins, arabinans, galactans andarabinogalactans. Pectins are the most complex polysaccharides in theplant cell wall. They are built up around a core chain of α(1,4)-linkedD-galacturonic acid units interspersed to some degree with L-rhamnose.In any one cell wall there are a number of structural units that fitthis description and it has generally been considered that in a singlepectic molecule, the core chains of different structural units arecontinuous with one another. The principal types of structural unit are:galacturonan (homogalacturonan), which may be substituted with methanolon the carboxyl group and acetate on O-2 and O-3; rhamnogalacturonan I(RGI), in which galacturonic acid units alternate with rhamnose unitscarrying (1,4)-linked galactan and (1,5)-linked arabinan side-chains.The arabinan side-chains may be attached directly to rhamnose orindirectly through the galactan chains; xylogalacturonan, with singlexylosyl units on O-3 of galacturonic acid (closely associated with RGI);and rhamnogalacturonan II (RGII), a particularly complex minor unitcontaining unusual sugars, for example apiose. An RGII unit may containtwo apiosyl residues which, under suitable ionic conditions, canreversibly form esters with borate.

Enzymes for use in the integrated processes of the current inventioncomprise preferably at least two activities, although typically enzymeswill comprise more than two activities, for example, three, four, five,six, seven, eight, nine or even more activities. Typically, enzymes foruse in the integrated processes of the current invention comprise atleast two cellulases. The at least two cellulases may contain the sameor different activities. Enzymes for use in the integrated processes ofthe current invention may also comprises at least one enzyme other thana cellulase. Preferably, the at least one other enzyme has an auxiliaryenzyme activity, i.e. an additional activity which, either directly orindirectly leads to lignocellulose degradation. Examples of suchauxiliary activities are mentioned herein and include, but are notlimited to hemicellulases.

Thus, enzymes for use in the integrated processes of the currentinvention may comprise lytic polysaccharide monooxygenase activity,endoglucanase activity and/or cellobiohydrolase activity and/orbeta-glucosidase activity. Enzymes for use in the invention may comprisemore than one enzyme activity per activity class. For example, enzymesfor use in the invention may comprise two endoglucanase activities, forexample, endo-1,3(1,4)-β glucanase activity and endo-β-1,4-glucanaseactivity.

Enzymes for use in the integrated processes of the current invention maybe derived from a fungus, such as a filamentous fungus such asRasamsonia, such as Rasamsonia emersonii. In an embodiment a core set of(lignocellulose degrading) enzyme activities may be derived fromRasamsonia emersonii. Rasamsonia emersonii can provide a highlyeffective set of activities as demonstrated herein for the hydrolysis oflignocellulosic material. If needed, the set of activities can besupplemented with additional enzyme activities from other sources. Suchadditional activities may be derived from classical sources and/orproduced by a genetically modified organisms.

The enzyme activities for use in the integrated processes of the currentinvention may be thermostable. Herein, this means that the activity hasa temperature optimum of 60° C. or higher, 70° C. or higher, 75° C. orhigher, 80° C. or higher, 85° C. or higher. Activities for use in theintegrated processes of the current invention will typically not havethe same temperature optima, but preferably will, nevertheless, bethermostable.

In addition, enzyme activities for use in the integrated processes ofthe current invention may be able to work at low pH. For the purposes ofthis invention, low pH indicates a pH of 5.5 or lower, 5 or lower, 4.9or lower, 4.8 or lower, 4.7 or lower, 4.6 or lower, 4.5 or lower, 4.4 orlower, 4.3 or lower, 4.2 or lower, 4.1 or lower, 4.0 or lower 3.9 orlower, 3.8 or lower, 3.7 or lower, 3.6 or lower, 3.5 or lower.

Activities for use in the integrated processes of the current inventionmay be defined by a combination of any of the above temperature optimaand pH values.

Enzymes for use in the integrated processes of the current invention maycomprise a cellulase and/or a hemicellulase and/or a pectinase from asource other than Rasamsonia. They may be used together with one or moreRasamsonia enzymes or they may be used without additional Rasamsoniaenzymes being present.

For example, enzymes for use in the integrated processes of the currentinvention may comprise a beta-glucosidase (BG) from Aspergillus, such asAspergillus oryzae, such as the one disclosed in WO 02/095014 or thefusion protein having beta-glucosidase activity disclosed in WO2008/057637, or Aspergillus fumigatus, such as the one disclosed as SEQID NO:2 in WO 2005/047499 or SEQ ID NO:5 in WO 2014/130812 or anAspergillus fumigatus beta-glucosidase variant, such as one disclosed inWO 2012/044915, such as one with the following substitutions: F100D,S283G, N456E, F512Y (using SEQ ID NO: 5 in WO 2014/130812 fornumbering), or Aspergillus aculeatus, Aspergillus niger or Aspergilluskawachi. In another embodiment the beta-glucosidase is derived fromPenicillium, such as Penicillium brasilianum disclosed as SEQ ID NO:2 inWO 2007/019442, or from Trichoderma, such as Trichoderma reesei, such asones described in U.S. Pat. Nos. 6,022,725, 6,982,159, 7,045,332,7,005,289, US 2006/0258554 US 2004/0102619. In an embodiment even abacterial beta-glucosidase can be used. In another embodiment thebeta-glucosidase is derived from Thielavia terrestris (WO 2011/035029)or Trichophaea saccata (WO 2007/019442).

For example, enzymes for use in the integrated processes of the currentinvention may comprise an endoglucanase (EG) from Trichoderma, such asTrichoderma reesei; from Humicola, such as a strain of Humicolainsolens; from Aspergillus, such as Aspergillus aculeatus or Aspergilluskawachii; from Erwinia, such as Erwinia carotovara; from Fusarium, suchas Fusarium oxysporum; from Thielavia, such as Thielavia terrestris;from Humicola, such as Humicola grisea var. thermoidea or Humicolainsolens; from Melanocarpus, such as Melanocarpus albomyces; fromNeurospora, such as Neurospora crassa; from Myceliophthora, such asMyceliophthora thermophila; from Cladorrhinum, such as Cladorrhinumfoecundissimum and/or from Chrysosporium, such as a strain ofChrysosporium lucknowense. In an embodiment even a bacterialendoglucanase can be used including, but are not limited to,Acidothermus cellulolyticus endoglucanase (see WO 91/05039; WO 93/15186;U.S. Pat. No. 5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO00/70031, WO 05/093050); Thermobifida fusca endoglucanase III (see WO05/093050); and Thermobifida fusca endoglucanase V (see WO 05/093050).

For example, enzymes for use in the integrated processes of the currentinvention may comprise a cellobiohydrolase I from Aspergillus, such asAspergillus fumigatus, such as the Cel7A CBH I disclosed in SEQ ID NO:6in WO 2011/057140 or SEQ ID NO:6 in WO 2014/130812, or from Trichoderma,such as Trichoderma reesei.

For example, enzymes for use in the integrated processes of the currentinvention may comprise a cellobiohydrolase II from Aspergillus, such asAspergillus fumigatus, such as the one in SEQ ID NO:7 in WO 2014/130812or from Trichoderma, such as Trichoderma reesei, or from Thielavia, suchas Thielavia terrestris, such as cellobiohydrolase II CEL6A fromThielavia terrestris.

For example, enzymes for use in the integrated processes of the currentinvention may comprise a GH61 polypeptide (a lytic polysaccharidemonooxygenase) from Thermoascus, such as Thermoascus aurantiacus, suchas the one described in WO 2005/074656 as SEQ ID NO:2 and SEQ ID NO:1 inWO2014/130812 and in WO 2010/065830; or from Thielavia, such asThielavia terrestris, such as the one described in WO 2005/074647 as SEQID NO: 8 or SEQ ID NO:4 in WO2014/130812 and in WO 2008/148131, and WO2011/035027; or from Aspergillus, such as Aspergillus fumigatus, such asthe one described in WO 2010/138754 as SEQ ID NO:2 or SEQ ID NO: 3 inWO2014/130812; or from Penicillium, such as Penicillium emersonii, suchas the one disclosed as SEQ ID NO:2 in WO 2011/041397 or SEQ ID NO:2 inWO2014/130812. Other suitable GH61 polypeptides include, but are notlimited to, Trichoderma reesei (see WO 2007/089290), Myceliophthorathermophila (see WO 2009/085935, WO 2009/085859, WO 2009/085864, WO2009/085868), Penicillium pinophilum (see WO 2011/005867), Thermoascussp. (see WO 2011/039319), and Thermoascus crustaceous (see WO2011/041504). In one aspect, the GH61 polypeptide is used in thepresence of a soluble activating divalent metal cation according to WO2008/151043, e.g. manganese sulfate. In one aspect, the GH61 polypeptideis used in the presence of a dioxy compound, a bicyclic compound, aheterocyclic compound, a nitrogen-containing compound, a quinonecompound, a sulfur-containing compound, or a liquor obtained from apretreated cellulosic material such as pretreated corn stover.

Other cellulolytic enzymes that may be used in the integrated processesof the present invention are described in WO 98/13465, WO 98/015619, WO98/015633, WO 99/06574, WO 99/10481, WO 99/025847, WO 99/031255, WO2002/101078, WO 2003/027306, WO 2003/052054, WO 2003/052055, WO2003/052056, WO 2003/052057, WO 2003/052118, WO 2004/016760, WO2004/043980, WO 2004/048592, WO 2005/001065, WO 2005/028636, WO2005/093050, WO 2005/093073, WO 2006/074005, WO 2006/117432, WO2007/071818, WO 2007/071820, WO 2008/008070, WO 2008/008793, U.S. Pat.Nos. 5,457,046, 5,648,263, and 5,686,593, to name just a few.

In addition, examples of xylanases useful in the integrated processes ofthe present invention include, but are not limited to, xylanases fromAspergillus aculeatus (see WO 94/21785), Aspergillus fumigatus (see WO2006/078256), Penicillium pinophilum (see WO 2011/041405), Penicilliumsp. (see WO 2010/126772), Thielavia terrestris NRRL 8126 (see WO2009/079210), and Trichophaea saccata GH10 (see WO 2011/057083).Examples of beta-xylosidases useful in the integrated processes of thepresent invention include, but are not limited to, beta-xylosidases fromNeurospora crassa and Trichoderma reesei. Examples of acetylxylanesterases useful in the processes of the present invention include, butare not limited to, acetylxylan esterases from Aspergillus aculeatus(see WO 2010/108918), Chaetomium globosum, Chaetomium gracile, Humicolainsolens DSM 1800 (see WO 2009/073709), Hypocrea jecorina (see WO2005/001036), Myceliophtera thermophila (see WO 2010/014880), Neurosporacrassa, Phaeosphaeria nodorum and Thielavia terrestris NRRL 8126 (see WO2009/042846). Examples of feruloyl esterases (ferulic acid esterases)useful in the integrated processes of the present invention include, butare not limited to, feruloyl esterases form Humicola insolens DSM 1800(see WO 2009/076122), Neosartorya fischeri, Neurospora crassa,Penicillium aurantiogriseum (see WO 2009/127729), and Thielaviaterrestris (see WO 2010/053838 and WO 2010/065448). Examples ofarabinofuranosidases useful in the integrated processes of the presentinvention include, but are not limited to, arabinofuranosidases fromAspergillus niger, Humicola insolens DSM 1800 (see WO 2006/114094 and WO2009/073383) and M. giganteus (see WO 2006/114094). Examples ofalpha-glucuronidases useful in the processes of the present inventioninclude, but are not limited to, alpha-glucuronidases from Aspergillusclavatus, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus,Humicola insolens (see WO 2010/014706), Penicillium aurantiogriseum (seeWO 2009/068565) and Trichoderma reesei.

Enzymes for use in the integrated processes of the current invention maycomprise one, two, three, four classes or more of cellulase, for exampleone, two, three or four or all of a lytic polysaccharide monooxygenas(LPMO), an endoglucanase (EG), one or two exo-cellobiohydrolase (CBH)and a beta-glucosidase (BG). Enzymes for use in the integrated processesof the current invention may comprise two or more of any of theseclasses of cellulase.

Enzymes for use in the integrated processes of the current invention maycomprise one type of cellulase activity and/or hemicellulase activityand/or pectinase activity provided by enzymes as described herein and asecond type of cellulase activity and/or hemicellulase activity and/orpectinase activity provided by an additionalcellulase/hemicellulase/pectinase.

As used herein, a cellulase is any polypeptide which is capable ofdegrading or modifying cellulose. A polypeptide which is capable ofdegrading cellulose is one which is capable of catalyzing the process ofbreaking down cellulose into smaller units, either partially, forexample into cellodextrins, or completely into glucose monomers. Acellulase according to the invention may give rise to a mixed populationof cellodextrins and glucose monomers. Such degradation will typicallytake place by way of a hydrolysis reaction.

Lytic polysaccharide monooxygenases (LPMO) are recently classified byCAZy in family AA9 (Auxiliary Activity Family 9) or family AA10(Auxiliary Activity Family 10). As mentioned above, lytic polysaccharidemonooxygenases are able to open a crystalline glucan structure. Lyticpolysaccharide monooxygenases may also affect cello-oligosaccharides.GH61 (glycoside hydrolase family 61 or sometimes referred to EGIV)proteins are (lytic) oxygen-dependent polysaccharide monooxygenases(PMO's/LPMO's) according to the latest literature (see lsaksen et al.,Journal of Biological Chemistry, vol. 289, no. 5, pp. 2632-2642). PMOand LPMO are used herein interchangeably. Often in literature theseproteins are mentioned to enhance the action of cellulases onlignocellulose substrates. GH61 was originally classified asendoglucanase based on measurement of very weak endo-1,4-β-d-glucanaseactivity in one family member. The term “GH61” as used herein, is to beunderstood as a family of enzymes, which share common conserved sequenceportions and folding to be classified in family 61 of thewell-established CAZy GH classification system (www.cazy.org/GH61.html).The glycoside hydrolase family 61 is a member of the family of glycosidehydrolases EC 3.2.1. GH61 are recently now reclassified by CAZy infamily AA9 (Auxiliary Activity Family 9). GH61 is used herein as beingpart of the cellulases.

CBM33 (family 33 carbohydrate-binding module) is a lytic polysaccharidemonooxygenase (see lsaksen et al, Journal of Biological Chemistry, vol.289, no. 5, pp. 2632-2642), CAZy has recently reclassified CBM33 in AA10(Auxiliary Activity Family 10).

As used herein, a hemicellulase is any polypeptide which is capable ofdegrading or modifying hemicellulose. That is to say, a hemicellulasemay be capable of degrading or modifying one or more of xylan,glucuronoxylan, arabinoxylan, glucomannan and xyloglucan. A polypeptidewhich is capable of degrading a hemicellulose is one which is capable ofcatalyzing the process of breaking down the hemicellulose into smallerpolysaccharides, either partially, for example into oligosaccharides, orcompletely into sugar monomers, for example hexose or pentose sugarmonomers. A hemicellulase according to the invention may give rise to amixed population of oligosaccharides and sugar monomers. Suchdegradation will typically take place by way of a hydrolysis reaction.

As used herein, a pectinase is any polypeptide which is capable ofdegrading or modifying pectin. A polypeptide which is capable ofdegrading pectin is one which is capable of catalyzing the process ofbreaking down pectin into smaller units, either partially, for exampleinto oligosaccharides, or completely into sugar monomers. A pectinaseaccording to the invention may give rise to a mixed population ofoligosacchardies and sugar monomers. Such degradation will typicallytake place by way of a hydrolysis reaction.

Accordingly, enzymes for use in the integrated processes of the currentinvention may comprise any cellulase, for example, a lyticpolysaccharide monooxygenase (e.g. GH61), a cellobiohydrolase, anendo-β-1,4-glucanase, a beta-glucosidaseor a β-(1,3)(1,4)-glucanase.

As used herein, a cellobiohydrolase (EC 3.2.1.91) is any polypeptidewhich is capable of catalyzing the hydrolysis of 1,4-β-D-glucosidiclinkages in cellulose or cellotetraose, releasing cellobiose from theends of the chains. This enzyme may also be referred to as cellulase1,4-β-cellobiosidase, 1,4-β-cellobiohydrolase, 1,4-β-D-glucancellobiohydrolase, avicelase, exo-1,4-β-D-glucanase,exocellobiohydrolase or exoglucanase.

As used herein, an endo-β-1,4-glucanase (EC 3.2.1.4) is any polypeptidewhich is capable of catalyzing the endohydrolysis of 1,4-β-D-glucosidiclinkages in cellulose, lichenin or cereal β-D-glucans. Such apolypeptide may also be capable of hydrolyzing 1,4-linkages inβ-D-glucans also containing 1,3-linkages. This enzyme may also bereferred to as cellulase, avicelase, β-1,4-endoglucan hydrolase,β-1,4-glucanase, carboxymethyl cellulase, celludextrinase,endo-1,4-β-D-glucanase, endo-1,4-β-D-glucanohydrolase,endo-1,4-β-glucanase or endoglucanase.

As used herein, a beta-glucosidase (EC 3.2.1.21) is any polypeptidewhich is capable of catalysing the hydrolysis of terminal, non-reducingβ-D-glucose residues with release of β-D-glucose. Such a polypeptide mayhave a wide specificity for β-D-glucosides and may also hydrolyze one ormore of the following: a β-D-galactoside, an α-L-arabinoside, aβ-D-xyloside or a β-D-fucoside. This enzyme may also be referred to asamygdalase, β-D-glucoside glucohydrolase, cellobiase or gentobiase.

As used herein, a β-(1,3)(1,4)-glucanase (EC 3.2.1.73) is anypolypeptide which is capable of catalysing the hydrolysis of1,4-β-D-glucosidic linkages in β-D-glucans containing 1,3- and1,4-bonds. Such a polypeptide may act on lichenin and cerealβ-D-glucans, but not on β-D-glucans containing only 1,3- or 1,4-bonds.This enzyme may also be referred to as licheninase, 1,3-1,4-β-D-glucan4-glucanohydrolase, β-glucanase, endo-β-1,3-1,4 glucanase, lichenase ormixed linkage β-glucanase. An alternative for this type of enzyme is EC3.2.1.6, which is described as endo-1,3(4)-beta-glucanase. This type ofenzyme hydrolyses 1,3- or 1,4-linkages in beta-D-glucanse when theglucose residue whose reducing group is involved in the linkage to behydrolysed is itself substituted at C-3. Alternative names includeendo-1,3-beta-glucanase, laminarinase, 1,3-(1,3;1,4)-beta-D-glucan 3 (4)glucanohydrolase. Substrates include laminarin, lichenin and cerealbeta-D-glucans.

Enzymes for use in the integrated processes of the current invention maycomprise any hemicellulase, for example, an endoxylanase, aβ-xylosidase, a α-L-arabionofuranosidase, an α-D-glucuronidase, anacetyl xylan esterase, a feruloyl esterase, a coumaroyl esterase, anα-galactosidase, a β-galactosidase, a β-mannanase or a β-mannosidase.

As used herein, an endoxylanase (EC 3.2.1.8) is any polypeptide which iscapable of catalysing the endohydrolysis of 1,4-β-D-xylosidic linkagesin xylans. This enzyme may also be referred to as endo-1,4-β-xylanase or1,4-β-D-xylan xylanohydrolase. An alternative is EC 3.2.1.136, aglucuronoarabinoxylan endoxylanase, an enzyme that is able to hydrolyze1,4 xylosidic linkages in glucuronoarabinoxylans.

As used herein, a β-xylosidase (EC 3.2.1.37) is any polypeptide which iscapable of catalysing the hydrolysis of 1,4-β-D-xylans, to removesuccessive D-xylose residues from the non-reducing termini. Such enzymesmay also hydrolyze xylobiose. This enzyme may also be referred to asxylan 1,4-β-xylosidase, 1,4-β-D-xylan xylohydrolase,exo-1,4-β-xylosidase or xylobiase.

As used herein, an α-L-arabinofuranosidase (EC 3.2.1.55) is anypolypeptide which is capable of acting on α-L-arabinofuranosides,α-L-arabinans containing (1,2) and/or (1,3)- and/or (1,5)-linkages,arabinoxylans and arabinogalactans. This enzyme may also be referred toas α-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

As used herein, an α-D-glucuronidase (EC 3.2.1.139) is any polypeptidewhich is capable of catalysing a reaction of the following form:alpha-D-glucuronoside+H(2)O=an alcohol+D-glucuronate. This enzyme mayalso be referred to as alpha-glucuronidase or alpha-glucosiduronase.These enzymes may also hydrolyse 4-O-methylated glucoronic acid, whichcan also be present as a substituent in xylans. An alternative is EC3.2.1.131: xylan alpha-1,2-glucuronosidase, which catalyses thehydrolysis of alpha-1,2-(4-O-methyl)glucuronosyl links.

As used herein, an acetyl xylan esterase (EC 3.1.1.72) is anypolypeptide which is capable of catalysing the deacetylation of xylansand xylo-oligosaccharides. Such a polypeptide may catalyze thehydrolysis of acetyl groups from polymeric xylan, acetylated xylose,acetylated glucose, alpha-napthyl acetate or p-nitrophenyl acetate but,typically, not from triacetylglycerol. Such a polypeptide typically doesnot act on acetylated mannan or pectin.

As used herein, a feruloyl esterase (EC 3.1.1.73) is any polypeptidewhich is capable of catalysing a reaction of the form:feruloyl-saccharide+H₂O=ferulate+saccharide. The saccharide may be, forexample, an oligosaccharide or a polysaccharide. It may typicallycatalyse the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl)group from an esterified sugar, which is usually arabinose in ‘natural’substrates. p-nitrophenol acetate and methyl ferulate are typicallypoorer substrates. This enzyme may also be referred to as cinnamoylester hydrolase, ferulic acid esterase or hydroxycinnamoyl esterase. Itmay also be referred to as a hemicellulase accessory enzyme, since itmay help xylanases and pectinases to break down plant cell wallhemicellulose and pectin.

As used herein, a coumaroyl esterase (EC 3.1.1.73) is any polypeptidewhich is capable of catalysing a reaction of the form:coumaroyl-saccharide+H(2)O=coumarate+saccharide. The saccharide may be,for example, an oligosaccharide or a polysaccharide. This enzyme mayalso be referred to as trans-4-coumaroyl esterase, trans-p-coumaroylesterase, p-coumaroyl esterase or p-coumaric acid esterase. This enzymealso falls within EC 3.1.1.73 so may also be referred to as a feruloylesterase.

As used herein, an α-galactosidase (EC 3.2.1.22) is any polypeptidewhich is capable of catalysing the hydrolysis of terminal, non-reducingα-D-galactose residues in α-D-galactosides, including galactoseoligosaccharides, galactomannans, galactans and arabinogalactans. Such apolypeptide may also be capable of hydrolyzing α-D-fucosides. Thisenzyme may also be referred to as melibiase.

As used herein, a β-galactosidase (EC 3.2.1.23) is any polypeptide whichis capable of catalysing the hydrolysis of terminal non-reducingβ-D-galactose residues in β-D-galactosides. Such a polypeptide may alsobe capable of hydrolyzing α-L-arabinosides. This enzyme may also bereferred to as exo-(1->4)-β-D-galactanase or lactase.

As used herein, a β-mannanase (EC 3.2.1.78) is any polypeptide which iscapable of catalysing the random hydrolysis of 1,4-β-D-mannosidiclinkages in mannans, galactomannans and glucomannans. This enzyme mayalso be referred to as mannan endo-1,4-β-mannosidase orendo-1,4-mannanase.

As used herein, a β-mannosidase (EC 3.2.1.25) is any polypeptide whichis capable of catalysing the hydrolysis of terminal, non-reducingβ-D-mannose residues in β-D-mannosides. This enzyme may also be referredto as mannanase or mannase.

Enzymes for use in the integrated processes of the current invention maycomprise any pectinase, for example an endo polygalacturonase, a pectinmethyl esterase, an endo-galactanase, a beta galactosidase, a pectinacetyl esterase, an endo-pectin lyase, pectate lyase, alpharhamnosidase, an exo-galacturonase, an expolygalacturonate lyase, arhamnogalacturonan hydrolase, a rhamnogalacturonan lyase, arhamnogalacturonan acetyl esterase, a rhamnogalacturonangalacturonohydrolase, a xylogalacturonase.

As used herein, an endo-polygalacturonase (EC 3.2.1.15) is anypolypeptide which is capable of catalysing the random hydrolysis of1,4-α-D-galactosiduronic linkages in pectate and other galacturonans.This enzyme may also be referred to as polygalacturonase pectindepolymerase, pectinase, endopolygalacturonase, pectolase, pectinhydrolase, pectin polygalacturonase, poly-α-1,4-galacturonideglycanohydrolase, endogalacturonase; endo-D-galacturonase orpoly(1,4-α-D-galacturonide) glycanohydrolase.

As used herein, a pectin methyl esterase (EC 3.1.1.11) is any enzymewhich is capable of catalysing the reaction: pectin+n H₂O=nmethanol+pectate. The enzyme may also been known as pectinesterase,pectin demethoxylase, pectin methoxylase, pectin methylesterase,pectase, pectinoesterase or pectin pectylhydrolase.

As used herein, an endo-galactanase (EC 3.2.1.89) is any enzyme capableof catalysing the endohydrolysis of 1,4-β-D-galactosidic linkages inarabinogalactans. The enzyme may also be known as arabinogalactanendo-1,4-β-galactosidase, endo-1,4-β-galactanase, galactanase,arabinogalactanase or arabinogalactan 4-β-D-galactanohydrolase.

As used herein, a pectin acetyl esterase is defined herein as any enzymewhich has an acetyl esterase activity which catalyses the deacetylationof the acetyl groups at the hydroxyl groups of GalUA residues of pectin.

As used herein, an endo-pectin lyase (EC 4.2.2.10) is any enzyme capableof catalysing the eliminative cleavage of (1-4)-α-D-galacturonan methylester to give oligosaccharides with4-deoxy-6-O-methyl-α-D-galact-4-enuronosyl groups at their non-reducingends. The enzyme may also be known as pectin lyase, pectintrans-eliminase; endo-pectin lyase, polymethylgalacturonictranseliminase, pectin methyltranseliminase, pectolyase, PL, PNL or PMGLor (1→4)-6-O-methyl-α-D-galacturonan lyase.

As used herein, a pectate lyase (EC 4.2.2.2) is any enzyme capable ofcatalysing the eliminative cleavage of (1→4)-α-D-galacturonan to giveoligosaccharides with 4-deoxy-α-D-galact-4-enuronosyl groups at theirnon-reducing ends. The enzyme may also be known polygalacturonictranseliminase, pectic acid transeliminase, polygalacturonate lyase,endopectin methyltranseliminase, pectate transeliminase,endogalacturonate transeliminase, pectic acid lyase, pectic lyase,α-1,4-D-endopolygalacturonic acid lyase, PGA lyase, PPase-N,endo-α-1,4-polygalacturonic acid lyase, polygalacturonic acid lyase,pectin trans-eliminase, polygalacturonic acid trans-eliminase or(1→4)-α-D-galacturonan lyase.

As used herein, an alpha rhamnosidase (EC 3.2.1.40) is any polypeptidewhich is capable of catalysing the hydrolysis of terminal non-reducingα-L-rhamnose residues in α-L-rhamnosides or alternatively inrhamnogalacturonan. This enzyme may also be known as α-L-rhamnosidase T,α-L-rhamnosidase N or α-L-rhamnoside rhamnohydrolase.

As used herein, exo-galacturonase (EC 3.2.1.82) is any polypeptidecapable of hydrolysis of pectic acid from the non-reducing end,releasing digalacturonate. The enzyme may also be known asexo-poly-α-galacturonosidase, exopolygalacturonosidase orexopolygalacturanosidase.

As used herein, exo-galacturonase (EC 3.2.1.67) is any polypeptidecapable of catalysing:(1,4-α-D-galacturonide)_(n)+H₂O=(1,4-α-D-galacturonide)_(n-1)+D-galacturonate.The enzyme may also be known as galacturan 1,4-α-galacturonidase,exopolygalacturonase, poly(galacturonate) hydrolase,exo-D-galacturonase, exo-D-galacturonanase, exopoly-D-galacturonase orpoly(1,4-α-D-galacturonide) galacturonohydrolase.

As used herein, exopolygalacturonate lyase (EC 4.2.2.9) is anypolypeptide capable of catalysing eliminative cleavage of4-(4-deoxy-α-D-galact-4-enuronosyl)-D-galacturonate from the reducingend of pectate, i.e. de-esterified pectin. This enzyme may be known aspectate disaccharide-lyase, pectate exo-lyase, exopectic acidtranseliminase, exopectate lyase, exopolygalacturonicacid-trans-eliminase, PATE, exo-PATE, exo-PGL or (1→4)-α-D-galacturonanreducing-end-disaccharide-lyase.

As used herein, rhamnogalacturonan hydrolase is any polypeptide which iscapable of hydrolyzing the linkage between galactosyluronic acid andrhamnopyranosyl in an endo-fashion in strictly alternatingrhamnogalacturonan structures, consisting of the disaccharide[(1,2-alpha-L-rhamnoyl-(1,4)-alpha-galactosyluronic acid].

As used herein, rhamnogalacturonan lyase is any polypeptide which is anypolypeptide which is capable of cleaving α-L-Rhap-(1→4)-α-D-GalpAlinkages in an endo-fashion in rhamnogalacturonan by beta-elimination.

As used herein, rhamnogalacturonan acetyl esterase is any polypeptidewhich catalyzes the deacetylation of the backbone of alternatingrhamnose and galacturonic acid residues in rhamnogalacturonan.

As used herein, rhamnogalacturonan galacturonohydrolase is anypolypeptide which is capable of hydrolyzing galacturonic acid from thenon-reducing end of strictly alternating rhamnogalacturonan structuresin an exo-fashion.

As used herein, xylogalacturonase is any polypeptide which acts onxylogalacturonan by cleaving the p-xylose substituted galacturonic acidbackbone in an endo-manner. This enzyme may also be known asxylogalacturonan hydrolase.

As used herein, an α-L-arabinofuranosidase (EC 3.2.1.55) is anypolypeptide which is capable of acting on α-L-arabinofuranosides,α-L-arabinans containing (1,2) and/or (1,3)- and/or (1,5)-linkages,arabinoxylans and arabinogalactans. This enzyme may also be referred toas α-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

As used herein, endo-arabinanase (EC 3.2.1.99) is any polypeptide whichis capable of catalysing endohydrolysis of 1,5-α-arabinofuranosidiclinkages in 1,5-arabinans. The enzyme may also be known asendo-arabinase, arabinan endo-1,5-α-L-arabinosidase,endo-1,5-α-L-arabinanase, endo-α-1,5-arabanase; endo-arabanase or1,5-α-L-arabinan 1,5-α-L-arabinanohydrolase.

Enzymes for use in the integrated processes of the current inventionwill typically comprise at least two cellulases and optionally at leastone hemicellulase and optionally at least one pectinase. Enzymes for usein the integrated processes of the current invention may comprise alytic polysaccharide monooxygenases (such as GH61), a cellobiohydrolase,an endoglucanase and/or a beta-glucosidase. Such enzymes may alsocomprise one or more hemicellulases and/or one or more pectinases.

In addition, one or more (for example two, three, four or all) of anamylase, a protease, a lipase, a ligninase, a hexosyltransferase, aglucuronidase, an expansin, a cellulose induced protein or a celluloseintegrating protein or like protein may be present in the enzymes foruse in the integrated processes of the current invention (these arereferred to as auxiliary activities above).

“Protease” includes enzymes that hydrolyze peptide bonds (peptidases),as well as enzymes that hydrolyze bonds between peptides and othermoieties, such as sugars (glycopeptidases). Many proteases arecharacterized under EC 3.4 and are suitable for use in the processes ofthe current invention. Some specific types of proteases include,cysteine proteases including pepsin, papain and serine proteasesincluding chymotrypsins, carboxypeptidases and metal loendopeptidases.

“Lipase” includes enzymes that hydrolyze lipids, fatty acids, andacylglycerides, including phospoglycerides, lipoproteins,diacylglycerols, and the like. In plants, lipids are used as structuralcomponents to limit water loss and pathogen infection. These lipidsinclude waxes derived from fatty acids, as well as cutin and suberin.

“Ligninase” includes enzymes that can hydrolyze or break down thestructure of lignin polymers. Enzymes that can break down lignin includelignin peroxidases, manganese peroxidases, laccases and feruloylesterases, and other enzymes described in the art known to depolymerizeor otherwise break lignin polymers. Also included are enzymes capable ofhydrolyzing bonds formed between hemicellulosic sugars (notablyarabinose) and lignin. Ligninases include but are not limited to thefollowing group of enzymes: lignin peroxidases (EC 1.11.1.14), manganeseperoxidases (EC 1.11.1.13), laccases (EC 1.10.3.2) and feruloylesterases (EC 3.1.1.73).

“Hexosyltransferase” (2.4.1-) includes enzymes which are capable ofcatalysing a transferase reaction, but which can also catalyze ahydrolysis reaction, for example of cellulose and/or cellulosedegradation products. An example of a hexosyltransferase which may beused in the invention is a β-glucanosyltransferase. Such an enzyme maybe able to catalyze degradation of (1,3)(1,4)glucan and/or celluloseand/or a cellulose degradation product.

“Glucuronidase” includes enzymes that catalyze the hydrolysis of aglucoronoside, for example β-glucuronoside to yield an alcohol. Manyglucuronidases have been characterized and may be suitable for use inthe invention, for example β-glucuronidase (EC 3.2.1.31),hyalurono-glucuronidase (EC 3.2.1.36), glucuronosyl-disulfoglucosamineglucuronidase (3.2.1.56), glycyrrhizinate β-glucuronidase (3.2.1.128) orα-D-glucuronidase (EC 3.2.1.139).

Enzymes for use in the integrated processes of the current invention maycomprise an expansin or expansin-like protein, such as a swollenin (seeSalheimo et al., Eur. J. Biochem. 269, 4202-4211, 2002) or aswollenin-like protein.

Expansins are implicated in loosening of the cell wall structure duringplant cell growth. Expansins have been proposed to disrupt hydrogenbonding between cellulose and other cell wall polysaccharides withouthaving hydrolytic activity. In this way, they are thought to allow thesliding of cellulose fibers and enlargement of the cell wall. Swollenin,an expansin-like protein contains an N-terminal Carbohydrate BindingModule Family 1 domain (CBD) and a C-terminal expansin-like domain. Forthe purposes of this invention, an expansin-like protein orswollenin-like protein may comprise one or both of such domains and/ormay disrupt the structure of cell walls (such as disrupting cellulosestructure), optionally without producing detectable amounts of reducingsugars.

Enzymes for use in the integrated processes of the current invention maycomprise a cellulose induced protein, for example the polypeptideproduct of the cip1 or cip2 gene or similar genes (see Foreman et al.,J. Biol. Chem. 278(34), 31988-31997, 2003), a cellulose/cellulosomeintegrating protein, for example the polypeptide product of the cipA orcipC gene, or a scaffoldin or a scaffoldin-like protein. Scaffoldins andcellulose integrating proteins are multi-functional integrating subunitswhich may organize cellulolytic subunits into a multi-enzyme complex.This is accomplished by the interaction of two complementary classes ofdomain, i.e. a cohesion domain on scaffoldin and a dockerin domain oneach enzymatic unit. The scaffoldin subunit also bears acellulose-binding module (CBM) that mediates attachment of thecellulosome to its substrate. A scaffoldin or cellulose integratingprotein for the purposes of this invention may comprise one or both ofsuch domains.

Enzymes for use in the integrated processes of the current invention mayalso comprise a catalase. The term “catalase” means a hydrogen-peroxide:hydrogen-peroxide oxidoreductase (EC 1.11.1.6 or EC 1.11.1.21) thatcatalyzes the conversion of two hydrogen peroxides to oxygen and twowaters. Catalase activity can be determined by monitoring thedegradation of hydrogen peroxide at 240 nm based on the followingreaction: 2H₂O₂→2H₂O+O₂. The reaction is conducted in 50 mM phosphate pH7.0 at 25° C. with 10.3 mM substrate (H₂0₂) and approximately 100 unitsof enzyme per ml. Absorbance is monitored spectrophotometrically within16-24 seconds, which should correspond to an absorbance reduction from0.45 to 0.4. One catalase activity unit can be expressed as onemicromole of H₂0₂ degraded per minute at pH 7.0 and 25° C.

Enzymes for use in the integrated processes of the current invention maybe composed of a member of each of the classes of enzymes mentionedabove, several members of one enzyme class, or any combination of theseenzymes classes or helper proteins (i.e. those proteins mentioned hereinwhich do not have enzymatic activity per se, but do nevertheless assistin lignocellulosic degradation).

Enzymes for use in the integrated processes of the current invention maybe composed of enzymes from (1) commercial suppliers; (2) cloned genesexpressing enzymes; (3) broth (such as that resulting from growth of amicrobial strain in media, wherein the strains secrete proteins andenzymes into the media; (4) cell lysates of strains grown as in (3);and/or (5) plant material expressing enzymes. Different enzymes may beobtained from different sources.

The enzymes can be produced either exogenously in microorganisms,yeasts, fungi, bacteria or plants, then isolated and added, for example,to (pretreated) lignocellulosic material. Alternatively, the enzyme maybe produced in a fermentation that uses (pretreated) lignocellulosicmaterial (such as corn stover or wheat straw) to provide nutrition to anorganism that produces an enzyme(s). In this manner, plants that producethe enzymes may themselves serve as a lignocellulosic material and beadded into lignocellulosic material.

In the uses and processes described herein, the enzymes described abovemay be provided concomitantly (i.e. in a single composition of enzymes)or separately or sequentially.

In an embodiment the enzymes are in the form of a whole fermentationbroth. The whole fermentation broth can be prepared from fermentation ofnon-recombinant and/or recombinant filamentous fungi. In an embodimentthe filamentous fungus is a recombinant filamentous fungus comprisingone or more genes which can be homologous or heterologous to thefilamentous fungus. In an embodiment, the filamentous fungus is arecombinant filamentous fungus comprising one or more genes which can behomologous or heterologous to the filamentous fungus wherein the one ormore genes encode enzymes that can degrade a cellulosic substrate. Thewhole fermentation broth may comprise any of the polypeptides or anycombination thereof.

Preferably, the composition of enzymes is whole fermentation brothwherein the cells are killed. The whole fermentation broth may containorganic acid(s) (used for killing the cells), killed cells and/or celldebris, and culture medium.

Generally, the filamentous fungi is cultivated in a cell culture mediumsuitable for production of enzymes capable of hydrolyzing a cellulosicsubstrate. The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable culture media, temperature rangesand other conditions suitable for growth and cellulase and/orhemicellulase and/or pectinase production are known in the art. Thewhole fermentation broth can be prepared by growing the filamentousfungi to stationary phase and maintaining the filamentous fungi underlimiting carbon conditions for a period of time sufficient to expressthe one or more cellulases and/or hemicellulases and/or pectinases. Onceenzymes, such as cellulases and/or hemicellulases and/or pectinases, aresecreted by the filamentous fungi into the fermentation medium, thewhole fermentation broth can be used. The whole fermentation broth ofthe present invention may comprise filamentous fungi. In someembodiments, the whole fermentation broth comprises the unfractionatedcontents of the fermentation materials derived at the end of thefermentation. Typically, the whole fermentation broth comprises thespent culture medium and cell debris present after the filamentous fungiis grown to saturation, incubated under carbon-limiting conditions toallow protein synthesis (particularly, expression of cellulases and/orhemicellulases and/or pectinases). In some embodiments, the wholefermentation broth comprises the spent cell culture medium,extracellular enzymes and filamentous fungi. In some embodiments, thefilamentous fungi present in whole fermentation broth can be lysed,permeabilized, or killed using methods known in the art to produce acell-killed whole fermentation broth. In an embodiment, the wholefermentation broth is a cell-killed whole fermentation broth, whereinthe whole fermentation broth containing the filamentous fungi cells arelysed or killed. In some embodiments, the cells are killed by lysing thefilamentous fungi by chemical and/or pH treatment to generate thecell-killed whole broth of a fermentation of the filamentous fungi. Insome embodiments, the cells are killed by lysing the filamentous fungiby chemical and/or pH treatment and adjusting the pH of the cell-killedfermentation mix to a suitable pH. In an embodiment, the wholefermentation broth comprises a first organic acid component comprisingat least one 1-5 carbon organic acid and/or a salt thereof and a secondorganic acid component comprising at least 6 or more carbon organic acidand/or a salt thereof. In an embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or any combination thereof and the second organic acid component isbenzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid,phenylacetic acid, a salt thereof, or any combination thereof.

The term “whole fermentation broth” as used herein refers to apreparation produced by cellular fermentation that undergoes no orminimal recovery and/or purification. For example, whole fermentationbroths are produced when microbial cultures are grown to saturation,incubated under carbon-limiting conditions to allow protein synthesis(e.g., expression of enzymes by host cells) and secretion into cellculture medium. Typically, the whole fermentation broth isunfractionated and comprises spent cell culture medium, extracellularenzymes, and microbial, preferably non-viable, cells.

If needed, the whole fermentation broth can be fractionated and the oneor more of the fractionated contents can be used. For instance, thekilled cells and/or cell debris can be removed from a whole fermentationbroth to provide a composition that is free of these components.

The whole fermentation broth may further comprise a preservative and/oranti-microbial agent. Such preservatives and/or agents are known in theart.

The whole fermentation broth as described herein is typically a liquid,but may contain insoluble components, such as killed cells, cell debris,culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedwhole fermentation broth.

In an embodiment, the whole fermentation broth may be supplemented withone or more enzyme activities that are not expressed endogenously, orexpressed at relatively low level by the filamentous fungi, to improvethe degradation of the cellulosic substrate, for example, to fermentablesugars such as glucose or xylose. The supplemental enzyme(s) can beadded as a supplement to the whole fermentation broth and the enzymesmay be a component of a separate whole fermentation broth, or may bepurified, or minimally recovered and/or purified.

In an embodiment, the whole fermentation broth comprises a wholefermentation broth of a fermentation of a recombinant filamentous fungioverexpressing one or more enzymes to improve the degradation of thecellulosic substrate. Alternatively, the whole fermentation broth cancomprise a mixture of a whole fermentation broth of a fermentation of anon-recombinant filamentous fungus and a recombinant filamentous fungusoverexpressing one or more enzymes to improve the degradation of thecellulosic substrate. In an embodiment, the whole fermentation brothcomprises a whole fermentation broth of a fermentation of a filamentousfungi overexpressing beta-glucosidase. Alternatively, the wholefermentation broth for use in the present methods and reactivecompositions can comprise a mixture of a whole fermentation broth of afermentation of a non-recombinant filamentous fungus and a wholefermentation broth of a fermentation of a recombinant filamentous fungioverexpressing a beta-glucosidase.

Enzymes are present in the liquefaction step and in the saccharificationstep of the enzymatic hydrolysis. These enzymes may be the same or maybe different. Furthermore, as described above, additional enzymes areadded during the liquefaction step and the saccharification step of theintegrated processes according to the present invention. The enzymesadded may be enzymes that are already present in the liquefaction stepand in the saccharification step. Alternatively, they may be differentenzymes. Moreover, the additional enzymes added during the liquefactionstep may differ or may be the same as the additional enzymes addedduring the saccharification step of the integrated processes accordingto the present invention.

Lignocellulosic material as used herein includes any lignocellulosicand/or hemicellulosic material. Lignocellulosic material suitable foruse in the processes of the current invention includes biomass, e.g.virgin biomass and/or non-virgin biomass such as agricultural biomass,commercial organics, construction and demolition debris, municipal solidwaste, waste paper and yard waste. Common forms of biomass includetrees, shrubs and grasses, wheat, wheat straw, sugar cane, cane straw,sugar cane bagasse, switch grass, miscanthus, energy cane, corn, cornstover, corn husks, corn cobs, canola stems, soybean stems, sweetsorghum, corn kernel including fiber from kernels, products andby-products from milling of grains such as corn, wheat and barley(including wet milling and dry milling) often called “bran or fibre” aswell as municipal solid waste, waste paper and yard waste. The biomasscan also be, but is not limited to, herbaceous material, agriculturalresidues, forestry residues, municipal solid wastes, waste paper, andpulp and paper mill residues. “Agricultural biomass” includes branches,bushes, canes, corn and corn husks, energy crops, forests, fruits,flowers, grains, grasses, herbaceous crops, leaves, bark, needles, logs,roots, saplings, short rotation woody crops, shrubs, switch grasses,trees, vegetables, fruit peels, vines, sugar beet pulp, wheat midlings,oat hulls, and hard and soft woods (not including woods with deleteriousmaterials). In addition, agricultural biomass includes organic wastematerials generated from agricultural processes including farming andforestry activities, specifically including forestry wood waste.Agricultural biomass may be any of the aforementioned singularly or inany combination or mixture thereof. In a preferred embodiment thelignocellulosic material is sugar cane bagasse or sugar cane straw.

Cellulose is an organic compound with the formula (C₆H₁₀O₅)_(n), apolysaccharide consisting of a linear chain of several hundred to overten thousand β(1→4) linked D-glucose units. A glucan molecule is apolysaccharide of D-glucose monomers linked by glycosidic bonds. Hereinglucan and cellulose are used interchangeably for a polysaccharide ofD-glucose monomers linked by glycosidic bonds. Methods for thequantitative analysis of glucan or polysaccharide compositions arewell-known and described in the art and are for example summarized inCarvalho de Souza et al., Carbohydrate Polymers 95 (2013) 657-663. Ingeneral, 50 to 70% of the glucan is crystalline cellulose, the remainderis amorphous cellulose.

In an embodiment the lignocellulosic material is pretreated beforeand/or during the enzymatic hydrolysis. Pretreatment methods are knownin the art and include, but are not limited to, heat, mechanical,chemical modification, biological modification and any combinationthereof. Pretreatment is typically performed in order to enhance theaccessibility of the lignocellulosic material to enzymatic hydrolysisand/or hydrolyse the hemicellulose and/or solubilize the hemicelluloseand/or cellulose and/or lignin, in the lignocellulosic material. In anembodiment, the pretreatment comprises treating the lignocellulosicmaterial with steam explosion, hot water treatment or treatment withdilute acid or dilute base. Examples of pretreatment methods include,but are not limited to, steam treatment (e.g. treatment at 100-260° C.,at a pressure of 7-45 bar, at neutral pH, for 1-10 minutes), dilute acidtreatment (e.g. treatment with 0.1-5% H₂SO₄ and/or SO₂ and/or HNO₃and/or HCl, in presence or absence of steam, at 120-200° C., at apressure of 2-15 bar, at acidic pH, for 2-30 minutes), organosolvtreatment (e.g. treatment with 1-1.5% H₂SO₄ in presence of organicsolvent and steam, at 160-200° C., at a pressure of 7-30 bar, at acidicpH, for 30-60 minutes), lime treatment (e.g. treatment with 0.1-2%NaOH/Ca(OH)₂ in the presence of water/steam at 60-160° C., at a pressureof 1-10 bar, at alkaline pH, for 60-4800 minutes), ARP treatment (e.g.treatment with 5-15% NH₃, at 150-180° C., at a pressure of 9-17 bar, atalkaline pH, for 10-90 minutes), AFEX treatment (e.g. treatmentwith >15% NH₃, at 60-140° C., at a pressure of 8-20 bar, at alkaline pH,for 5-30 minutes).

The lignocellulosic material may be washed. In an embodiment thelignocellulosic material may be washed before and/or after thepretreatment. The washing step may be performed before and/or aftersolid/liquid separation of the lignocellulosic material and/or thepretreated lignocellulosic material. If performed after the solid/liquidseparation, the solid fraction obtained after solid/liquid separationmay be washed. The washing step may be used to remove water solublecompounds that may act as inhibitors for the fermentation and/orhydrolysis step. The washing step may be conducted in manner known tothe skilled person. Next to washing, other detoxification methods doexist. The pretreated lignocellulosic material may also be detoxified byany (or any combination) of these methods which include, but are notlimited to, solid/liquid separation, vacuum evaporation, extraction,adsorption, neutralization, overliming, addition of reducing agents,addition of detoxifying enzymes such as laccases or peroxidases,addition of microorganisms capable of detoxification of hydrolysates.

The enzymes used in the integrated processes of the invention canextremely effectively hydrolyze lignocellulosic material, for examplecorn stover, wheat straw, cane straw, and/or sugar cane bagasse, whichcan then be further converted into a product, such as ethanol, biogas,butanol, a plastic, an organic acid such as succinic acid, a solvent, ananimal feed supplement, a pharmaceutical, a vitamin, an amino acid, anenzyme or a chemical feedstock. Additionally, intermediate products froma process following the hydrolysis, for example lactic acid asintermediate in biogas production, can be used as building block forother materials. The present invention is exemplified with theproduction of ethanol and succinic acid, but this is done asexemplification only rather than as limitation, the other productsmentioned can be produced equally well.

In an embodiment the amount of enzyme added (herein also called enzymedosage or enzyme load) is low. In an embodiment the amount of enzyme is10 mg protein/g dry matter weight or lower, 9 mg protein/g dry matterweight or lower, 8 mg protein/g dry matter weight or lower, 7 mgprotein/g dry matter weight or lower, 6 mg protein/g dry matter weightor lower, 5 mg protein/g dry matter or lower, 4 mg protein/g dry matteror lower, 3 mg protein/g dry matter or lower, 2 mg protein/g dry matteror lower, or 1 mg protein/g dry matter or lower (expressed as protein inmg protein/g dry matter). In an embodiment, the amount of enzyme is 5 mgenzyme/g dry matter weight or lower, 4 mg enzyme/g dry matter weight orlower, 3 mg enzyme/g dry matter weight or lower, 2 mg enzyme/g drymatter weight or lower, 1 mg enzyme/g dry matter weight or lower, 0.5 mgenzyme/g dry matter weight or lower, 0.4 mg enzyme composition/g drymatter weight or lower, 0.3 mg enzyme/g dry matter weight or lower, 0.25mg enzyme/g dry matter weight or lower, 0.20 mg enzyme/g dry matterweight or lower, 0.18 mg enzyme/g dry matter weight or lower, 0.15 mgenzyme/g dry matter weight or lower or 0.10 mg enzyme/g dry matterweight or lower (expressed as total of cellulase enzymes in mg enzyme/gdry matter). A low enzyme dosage is possible, because of the activityand stability of the enzymes. When the enzymatic hydrolysis comprises aseparate liquefaction step and a saccharification step, enzyme may beadded before and/or during only one of the steps or before and/or duringboth steps.

The pH during the enzymatic hydrolysis may be chosen by the skilledperson. In an embodiment the pH during the hydrolysis may be 3.0 to 6.4.The stable enzymes of the invention may have a broad pH range of up to 2pH units, up to 3 pH units, up to 5 pH units. The optimum pH may liewithin the limits of pH 2.0 to 8.0, 2.5 to 7.5, 3.0 to 7.0, 3.5 to 6.5,4.0 to 5.0, 4.0 to 4.5 or is about 4.2. The pH used in the liquefactionstep of the enzymatic hydrolysis and the saccharification step of theenzymatic hydrolysis may differ or may be the same. In case differentenzymes are used during the liquefaction step and the saccharificationstep, the optimum pH of said enzymes may differ or may be the same.

In an embodiment the hydrolysis step is conducted until 70% or more, 80%or more, 85% or more, 90% or more, 92% or more, 95% or more of availablesugar in the lignocellulosic material is released.

Significantly, a process of the invention may be carried out using highlevels of dry matter (of the lignocellulosic material) in the hydrolysisreaction. In an embodiment the dry matter content at the end of theenzymatic hydrolysis is 5 wt % or higher, 6 wt % or higher, 7 wt % orhigher, 8 wt % or higher, 9 wt % or higher, 10 wt % or higher, 11 wt %or higher, 12 wt % or higher, 13 wt % or higher, 14 wt % or higher, 15wt % or higher, 16 wt % or higher, 17 wt % or higher, 18 wt % or higher,19 wt % or higher, 20 wt % or higher, 21 wt % or higher, 22 wt % orhigher, 23 wt % or higher, 24 wt % or higher, 25 wt % or higher, 26 wt %or higher, 27 wt % or higher, 28 wt % or higher, 29 wt % or higher, 30wt % or higher, 31 wt % or higher, 32 wt % or higher, 33 wt % or higher,34 wt % or higher, 35 wt % or higher, 36 wt % or higher, 37 wt % orhigher, 38 wt % or higher or 39 wt % or higher. In an embodiment the drymatter content at the end of the enzymatic hydrolysis is between 5 wt%-40 wt %, 6 wt %-40 wt %, 7 wt %-40 wt %, 8 wt %-40 wt %, 9 wt %-40 wt%, 10 wt %-40 wt %, 11 wt %-40 wt %, 12 wt %-40 wt %, 13 wt %-40 wt %,14 wt %-40 wt %, 15 wt %-40 wt %, 16 wt %-40 wt %, 17 wt %-40 wt %, 18wt %-40 wt %, 19 wt %-40 wt %, 20 wt %-40 wt %, 21 wt %-40 wt %, 22 wt%-40 wt %, 23 wt %-40 wt %, 24 wt %-40 wt %, 25 wt %-40 wt %, 26 wt %-40wt %, 27 wt %-40 wt %, 28 wt %-40 wt %, 29 wt %-40 wt %, 30 wt %-40 wt%, 31 wt %-40 wt %, 32 wt %-40 wt %, 33 wt %-40 wt %, 34 wt %-40 wt %,35 wt %-40 wt %, 36 wt %-40 wt %, 37 wt %-40 wt %, 38 wt %-40 wt %, 39wt %-40 wt %.

In an embodiment the dry matter content at the end of the liquefactionstep of the enzymatic hydrolysis is 5 wt % or higher, 6 wt % or higher,7 wt % or higher, 8 wt % or higher, 9 wt % or higher, 10 wt % or higher,11 wt % or higher, 12 wt % or higher, 13 wt % or higher, 14 wt % orhigher, 15 wt % or higher, 16 wt % or higher, 17 wt % or higher, 18 wt %or higher, 19 wt % or higher, 20 wt % or higher, 21 wt % or higher, 22wt % or higher, 23 wt % or higher, 24 wt % or higher, 25 wt % or higher,26 wt % or higher, 27 wt % or higher, 28 wt % or higher, 29 wt % orhigher, 30 wt % or higher, 31 wt % or higher, 32 wt % or higher, 33 wt %or higher, 34 wt % or higher, 35 wt % or higher, 36 wt % or higher, 37wt % or higher, 38 wt % or higher or 39 wt % or higher. In an embodimentthe dry matter content at the end of the liquefaction step of theenzymatic hydrolysis is between 5 wt %-40 wt %, 6 wt %-40 wt %, 7 wt%-40 wt %, 8 wt %-40 wt %, 9 wt %-40 wt %, 10 wt %-40 wt %, 11 wt %-40wt %, 12 wt %-40 wt %, 13 wt %-40 wt %, 14 wt %-40 wt %, 15 wt %-40 wt%, 16 wt %-40 wt %, 17 wt %-40 wt %, 18 wt %-40 wt %, 19 wt %-40 wt %,20 wt %-40 wt %, 21 wt %-40 wt %, 22 wt %-40 wt %, 23 wt %-40 wt %, 24wt %-40 wt %, 25 wt %-40 wt %, 26 wt %-40 wt %, 27 wt %-40 wt %, 28 wt%-40 wt %, 29 wt %-40 wt %, 30 wt %-40 wt %, 31 wt %-40 wt %, 32 wt %-40wt %, 33 wt %-40 wt %, 34 wt %-40 wt %, 35 wt %-40 wt %, 36 wt %-40 wt%, 37 wt %-40 wt %, 38 wt %-40 wt %, 39 wt %-40 wt %.

In an embodiment the dry matter content at the end of thesaccharification step of the enzymatic hydrolysis is 5 wt % or higher, 6wt % or higher, 7 wt % or higher, 8 wt % or higher, 9 wt % or higher, 10wt % or higher, 11 wt % or higher, 12 wt % or higher, 13 wt % or higher,14 wt % or higher, 15 wt % or higher, 16 wt % or higher, 17 wt % orhigher, 18 wt % or higher, 19 wt % or higher, 20 wt % or higher, 21 wt %or higher, 22 wt % or higher, 23 wt % or higher, 24 wt % or higher, 25wt % or higher, 26 wt % or higher, 27 wt % or higher, 28 wt % or higher,29 wt % or higher, 30 wt % or higher, 31 wt % or higher, 32 wt % orhigher, 33 wt % or higher, 34 wt % or higher, 35 wt % or higher, 36 wt %or higher, 37 wt % or higher, 38 wt % or higher or 39 wt % or higher. Inan embodiment the dry matter content at the end of the saccharificationstep of the enzymatic hydrolysis is between 5 wt %-40 wt %, 6 wt %-40 wt%, 7 wt %-40 wt %, 8 wt %-40 wt %, 9 wt %-40 wt %, 10 wt %-40 wt %, 11wt %-40 wt %, 12 wt %-40 wt %, 13 wt %-40 wt %, 14 wt %-40 wt %, 15 wt%-40 wt %, 16 wt %-40 wt %, 17 wt %-40 wt %, 18 wt %-40 wt %, 19 wt %-40wt %, 20 wt %-40 wt %, 21 wt %-40 wt %, 22 wt %-40 wt %, 23 wt %-40 wt%, 24 wt %-40 wt %, 25 wt %-40 wt %, 26 wt %-40 wt %, 27 wt %-40 wt %,28 wt %-40 wt %, 29 wt %-40 wt %, 30 wt %-40 wt %, 31 wt %-40 wt %, 32wt %-40 wt %, 33 wt %-40 wt %, 34 wt %-40 wt %, 35 wt %-40 wt %, 36 wt%-40 wt %, 37 wt %-40 wt %, 38 wt %-40 wt %, 39 wt %-40 wt %.

In an embodiment the fermentation steps in the integrated processesaccording to the present invention are performed in one or morecontainers. In an embodiment the fermentation of the at least solidfraction and/or the at least liquid fraction by an alcohol producingmicroorganism to produce alcohol is performed in one or more containers.In an embodiment the fermentation of the at least liquid fraction and/orthe at least solid fraction by an organic acid producing microorganismto produce an organic acid is performed in one or more containers. Thefermentation of the at least solid fraction and/or the at least liquidfraction by an alcohol producing microorganism to produce alcohol can bedone in the same container(s) wherein the enzymatic hydrolysis isperformed. Alternatively, the fermentation of the at least solidfraction and/or the at least liquid fraction by an alcohol producingmicroorganism to produce alcohol and the fermentation of the at leastliquid fraction and/or the at least solid fraction by an organic acidproducing microorganism to produce an organic acid can be performed inone or more separate containers, but may also be done in one or more ofthe same containers.

In an embodiment the alcohol producing microorganism is able to fermentat least a C5 sugar and at least a C6 sugar. In an embodiment theorganic acid producing microorganism is able to ferment at least a C6sugar. In an embodiment the alcohol producing microorganism and theorganic acid producing microorganism are different microorganisms. Inanother embodiment the alcohol producing microorganism and the organicacid producing microorganism are the same microorganism, i.e. thealcohol producing microorganism is also able to produce organic acidsuch as succinic acid. In an embodiment the alcohol producingmicroorganism and/or the organic acid producing microorganism is ayeast.

In a further aspect, the invention thus includes fermentation processesin which a microorganism is used for the fermentation of a carbon sourcecomprising sugar(s), e.g. glucose, L-arabinose and/or xylose. The carbonsource may include any carbohydrate oligo- or polymer comprisingL-arabinose, xylose or glucose units, such as e.g. lignocellulose,xylans, cellulose, starch, arabinan and the like. For release of xyloseor glucose units from such carbohydrates, appropriate carbohydrases(such as xylanases, glucanases, amylases and the like) may be added tothe fermentation medium or may be produced by the modified host cell. Inthe latter case, the modified host cell may be genetically engineered toproduce and excrete such carbohydrases. An additional advantage of usingoligo- or polymeric sources of glucose is that it enables to maintain alow(er) concentration of free glucose during the fermentation, e.g. byusing rate-limiting amounts of the carbohydrases. This, in turn, willprevent repression of systems required for metabolism and transport ofnon-glucose sugars such as xylose. In a preferred process the modifiedhost cell ferments both the L-arabinose (optionally xylose) and glucose,preferably simultaneously in which case preferably a modified host cellis used which is insensitive to glucose repression to prevent diauxicgrowth. In addition to a source of L-arabinose, optionally xylose (andglucose) as carbon source, the fermentation medium will further comprisethe appropriate ingredient required for growth of the modified hostcell. Compositions of fermentation media for growth of microorganismssuch as yeasts or filamentous fungi are well known in the art.

The fermentation time may be shorter than in conventional fermentationat the same conditions, wherein part of the enzymatic hydrolysis stillhas to take part during fermentation. In one embodiment, thefermentation time is 100 hours or less, 90 hours or less, 80 hours orless, 70 hours or less, or 60 hours or less, for a sugar composition of50 g/I glucose and corresponding other sugars from the lignocellulosicmaterial (e.g. 50 g/I xylose, 35 g/I L-arabinose and 10 g/I galactose).For more dilute sugar compositions, the fermentation time maycorrespondingly be reduced. In an embodiment the fermentation time ofthe ethanol production step is between 10 and 50 hours for ethanol madeout of C6 sugars and between 20 and 100 hours for ethanol made out of C5sugars. In an embodiment the fermentation time of the succinic acidproduction step is between 20 and 70 hours.

The fermentation process may be an aerobic or an anaerobic fermentationprocess. An anaerobic fermentation process is herein defined as afermentation process run in the absence of oxygen or in whichsubstantially no oxygen is consumed, preferably less than 5, 2.5 or 1mmol/L/h, more preferably 0 mmol/L/h is consumed (i.e. oxygenconsumption is not detectable), and wherein organic molecules serve asboth electron donor and electron acceptors. In the absence of oxygen,NADH produced in glycolysis and biomass formation, cannot be oxidised byoxidative phosphorylation. To solve this problem many microorganisms usepyruvate or one of its derivatives as an electron and hydrogen acceptorthereby regenerating NAD⁺. Thus, in a preferred anaerobic fermentationprocess pyruvate is used as an electron (and hydrogen acceptor) and isreduced to fermentation products such as ethanol, lactic acid,3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane-diol,ethylene, glycerol, butanol, a β-lactam antibiotics and a cephalosporin.In a preferred embodiment, the fermentation process is anaerobic. Ananaerobic process is advantageous, since it is cheaper than aerobicprocesses: less special equipment is needed. Furthermore, anaerobicprocesses are expected to give a higher product yield than aerobicprocesses. Under aerobic conditions, usually the biomass yield is higherthan under anaerobic conditions. As a consequence, usually under aerobicconditions, the expected product yield is lower than under anaerobicconditions.

In another embodiment, the fermentation process is under oxygen-limitedconditions. More preferably, the fermentation process is aerobic andunder oxygen-limited conditions. An oxygen-limited fermentation processis a process in which the oxygen consumption is limited by the oxygentransfer from the gas to the liquid. The degree of oxygen limitation isdetermined by the amount and composition of the ingoing gas flow as wellas the actual mixing/mass transfer properties of the fermentationequipment used. Preferably, in a process under oxygen-limitedconditions, the rate of oxygen consumption is at least 5.5, morepreferably at least 6 and even more preferably at least 7 mmol/L/h.

In an embodiment the alcohol fermentation process is anaerobic, whilethe organic acid fermentation process is aerobic, but done underoxygen-limited conditions.

The fermentation process is preferably run at a temperature that isoptimal for the modified cell. Thus, for most yeasts or fungal cells,the fermentation process is performed at a temperature which is lessthan 42° C., preferably 38° C. or lower. For yeast or filamentous fungalhost cells, the fermentation process is preferably performed at atemperature which is lower than 35, 33, 30 or 28° C. and at atemperature which is higher than 20, 22, or 25° C. In an embodiment thealcohol fermentation step and the organic acid fermentation step areperformed between 25° C. and 35° C.

In an embodiment of the invention, the fermentations are conducted witha fermenting microorganism. In an embodiment of the invention, thealcohol (e.g. ethanol) fermentations of C5 sugars are conducted with aC5 fermenting microorganism. In an embodiment of the invention, thealcohol (e.g. ethanol) fermentations of C6 sugars are conducted with aC5 fermenting microorganism or a commercial C6 fermenting microorganism.Commercially available yeast suitable for ethanol production include,but are not limited to, BIOFERM™ AFT and XR (NABC—North AmericanBioproducts Corporation, GA, USA), ETHANOL RED™ yeast(Fermentis/Lesaffre, USA), FALI™ (Fleischmann's Yeast, USA), FERMIOL™(DSM Specialties), GERT STRAND™ (Gert Strand AB, Sweden), andSUPERSTART™ and THERMOSACC™ fresh yeast (Ethanol Technology, WI, USA).In an embodiment the fermentations are performed in one or morecontainers. In an embodiment the fermentations are performed in the oneor more fermentation containers. In an embodiment propagation of thealcohol producing microorganism and/or the organic acid producingmicroorganism by fermentation of the at least liquid fraction and/or theat least solid fraction is performed in one or more propagationcontainers. After propagation, the alcohol producing microorganismand/or the organic acid producing microorganism may be added to one ormore fermentation containers. Alternatively, the propagation of thealcohol producing microorganism and/or the organic acid producingmicroorganism is combined with the fermentation of the at least liquidfraction and/or the at least solid fraction by the alcohol producingmicroorganism and/or the organic acid producing microorganism to producealcohol and/or organic acid, respectively.

In an embodiment the alcohol producing microorganism is a microorganismthat is able to ferment at least one C5 sugar. Preferably, it also isable to ferment at least one C6 sugar. In an embodiment the inventionrelates to an integrated process comprising the production of ethanol,wherein the process comprises the step of fermenting a medium containingsugar(s) with a microorganism that is able to ferment at least one C5sugar.

In an embodiment the organic acid producing microorganism is amicroorganism that is able to ferment at least one C6 sugar. In anembodiment the invention relates to an integrated process for theproduction of succinic acid, wherein the process comprises the step offermenting a medium containing sugar(s) with a microorganism that isable to ferment at least one C6 sugar.

The alcohol producing microorganisms may be a prokaryotic or eukaryoticorganism. The microorganism used in the process may be a geneticallyengineered microorganism. Examples of suitable alcohol producingorganisms are yeasts, for instance Saccharomyces, e.g. Saccharomycescerevisiae, Saccharomyces pastorianus or Saccharomyces uvarum,Hansenula, lssatchenkia, e.g. lssatchenkia orientalis, Pichia, e.g.Pichia stipites or Pichia pastoris, Kluyveromyces, e.g. Kluyveromycesfagilis, Candida, e.g. Candida pseudotropicalis or Candidaacidothermophilum, Pachysolen, e.g. Pachysolen tannophilus or bacteria,for instance Lactobacillus, e.g. Lactobacillus lactis, Geobacillus,Zymomonas, e.g. Zymomonas mobilis, Clostridium, e.g. Clostridiumphytofermentans, Escherichia, e.g. E. coli, Klebsiella, e.g. Klebsiellaoxytoca. In an embodiment the microorganism that is able to ferment atleast one C5 sugar is a yeast. In an embodiment, the yeast is belongs tothe genus Saccharomyces, preferably of the species Saccharomycescerevisiae. The yeast, e.g. Saccharomyces cerevisiae, used in theprocesses according to the present invention is capable of convertinghexose (C6) sugars and pentose (C5) sugars. The yeast, e.g.Saccharomyces cerevisiae, used in the processes according to the presentinvention can anaerobically ferment at least one C6 sugar and at leastone C5 sugar. For example, the yeast is capable of using L-arabinose andxylose in addition to glucose anaerobically. In an embodiment, the yeastis capable of converting L-arabinose into L-ribulose and/or xylulose5-phosphate and/or into a desired fermentation product, for example intoethanol. Organisms, for example Saccharomyces cerevisiae strains, ableto produce ethanol from L-arabinose may be produced by modifying a hostyeast introducing the araA (L-arabinose isomerase), araB(L-ribuloglyoxalate) and araD (L-ribulose-5-P4-epimerase) genes from asuitable source. Such genes may be introduced into a host cell in orderthat it is capable of using arabinose. Such an approach is given isdescribed in WO2003/095627. araA, araB and araD genes from Lactobacillusplantarum may be used and are disclosed in WO2008/041840. The araA genefrom Bacillus subtilis and the araB and araD genes from Escherichia colimay be used and are disclosed in EP1499708. In another embodiment, araA,araB and araD genes may derived from of at least one of the genusClavibacter, Arthrobacter and/or Gramella, in particular one ofClavibacter michiganensis, Arthrobacter aurescens, and/or Gramellaforsetii, as disclosed in WO 2009011591. In an embodiment, the yeast mayalso comprise one or more copies of xylose isomerase gene and/or one ormore copies of xylose reductase and/or xylitol dehydrogenase.

The yeast may comprise one or more genetic modifications to allow theyeast to ferment xylose. Examples of genetic modifications areintroduction of one or more xylA-gene, XYL1 gene and XYL2 gene and/orXKS1-gene; deletion of the aldose reductase (GRE3) gene; overexpressionof PPP-genes TALI, TKL1, RPE1 and RKI1 to allow the increase of the fluxthrough the pentose phosphate pathway in the cell. Examples ofgenetically engineered yeast are described in EP1468093 and/orWO2006/009434.

An example of a suitable commercial yeast is RN1016 that is a xylose andglucose fermenting Saccharomyces cerevisiae strain from DSM, theNetherlands.

In an embodiment, the fermentation process for the production of ethanolis anaerobic. Anaerobic has already been defined earlier herein. Inanother preferred embodiment, the fermentation process for theproduction of ethanol is aerobic. In another preferred embodiment, thefermentation process for the production of ethanol is underoxygen-limited conditions, more preferably aerobic and underoxygen-limited conditions. Oxygen-limited conditions have already beendefined earlier herein.

The volumetric ethanol productivity is preferably at least 0.5, 1.0,1.5, 2.0, 2.5, 3.0, 5.0 or 10.0 g ethanol per litre per hour. Theethanol yield on L-arabinose and optionally xylose and/or glucose in theprocess preferably is at least 20, 25, 30, 35, 40, 45, 50, 60, 70, 80,90, 95 or 98%. The ethanol yield is herein defined as a percentage ofthe theoretical maximum yield, which, for glucose and L-arabinose andoptionally xylose is 0.51 g ethanol per g glucose or xylose.

In one aspect, the fermentation process leading to the production ofethanol, has several advantages by comparison to known ethanolfermentations processes: anaerobic processes are possible; oxygenlimited conditions are possible; higher ethanol yields and ethanolproduction rates can be obtained; the strain used may be able to useL-arabinose and optionally xylose.

Alternatively to the fermentation processes described above, at leasttwo distinct cells may be used, this means this process is aco-fermentation process. All preferred embodiments of the fermentationprocesses as described above are also preferred embodiments of thisco-fermentation process: identity of the fermentation product, identityof source of L-arabinose and source of xylose, conditions offermentation (aerobic or anaerobic conditions, oxygen-limitedconditions, temperature at which the process is being carried out,productivity of ethanol, yield of ethanol).

The organic acid producing microorganisms may be a prokaryotic oreukaryotic organism. The microorganism used in the process may be agenetically engineered microorganism. Examples of suitable organic acidproducing organisms are yeasts, for instance Saccharomyces, e.g.Saccharomyces cerevisiae; fungi for instance Aspergillus strains, suchas Aspergillus niger and Aspergillus fumigatus, Byssochlamys nivea,Lentinus degener, Paecilomyces varioti and Penicillium viniferum; andbacteria, for instance Anaerobiospirillum succiniciproducens,Actinobacillus succinogenes, Mannhei succiniciproducers MBEL 55E,Escherichia coli, Propionibacterium species, Pectinatus sp., Bacteroidessp., such as Bacteroides amylophilus, Ruminococcus flavefaciens,Prevotella ruminicola, Succcinimonas amylolytica, Succinivibriodextrinisolvens, Wolinella succinogenes, and Cytophaga succinicans. Inan embodiment the organic acid producing microorganism that is able toferment at least one C6 sugar is a yeast. In an embodiment, the yeast isbelongs to the genus Saccharomyces, preferably of the speciesSaccharomyces cerevisiae. The yeast, e.g. Saccharomyces cerevisiae, usedin the production processes of organic acid according to the presentinvention is capable of converting hexose (C6) sugars. The yeast, e.g.Saccharomyces cerevisiae, used in the processes according to the presentinvention can anaerobically ferment at least one C6 sugar.

The fermentation processes may be carried out without any requirement toadjust the pH during the processes. That is to say, the processes areones which may be carried out without the addition of any acid(s) orbase(s). However, this excludes a pretreatment step, where acid may beadded. The point is that the enzymes used in the processes of theinvention is capable of acting at low pH and, therefore, there is noneed to adjust the pH of acid of an acid pretreated feedstock in orderthat hydrolysis may take place. Accordingly, the processes of theinvention may be zero waste processes using only organic products withno requirement for inorganic chemical input.

The overall reaction time (or the reaction time of hydrolysis step andfermentation step together) may be reduced. In one embodiment, theoverall reaction time is 300 hours or less, 200 hours or less, 150 hoursor less, 140 hours or less, 130 or less, 120 hours or less, 110 hours orless, 100 hours of less, 90 hours or less, 80 hours or less, 75 hours orless, or about 72 hours at 90% glucose yield. Correspondingly, loweroverall reaction times may be reached at lower glucose yield.

Other fermentation products that may be produced by the integratedprocesses of the invention can be any substance derived fromfermentation. They include, but are not limited to, alcohol (such asarabinitol, butanol, ethanol, glycerol, methanol, 1,3-propanediol,sorbitol, and xylitol); organic acid (such as acetic acid, acetonicacid, adipic acid, ascorbic acid, acrylic acid, citric acid,2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid,gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid,itaconic acid, lactic acid, maleic acid, malic acid, malonic acid,oxalic acid, oxaloacetic acid, propionic acid, succinic acid, andxylonic acid); ketones (such as acetone); amino acids (such as asparticacid, glutamic acid, glycine, lysine, serine, tryptophan, andthreonine); alkanes (such as pentane, hexane, heptane, octane, nonane,decane, undecane, and dodecane), cycloalkanes (such as cyclopentane,cyclohexane, cycloheptane, and cyclooctane), alkenes (such as pentene,hexene, heptene, and octene); and gases (such as methane, hydrogen (H₂),carbon dioxide (C0₂), and carbon monoxide (CO)). The fermentationproduct can also be a protein, a vitamin, a pharmaceutical, an animalfeed supplement, a specialty chemical, a chemical feedstock, a plastic,a solvent, ethylene, an enzyme, such as a protease, a cellulase, anamylase, a glucanase, a lactase, a lipase, a lyase, an oxidoreductase, atransferase or a xylanase. In a preferred embodiment the organic acid issuccinic acid and/or the alcohol is ethanol.

In an embodiment the alcohol, the organic acid, the enzymes, the enzymeproducing microorganism, the alcohol producing microorganism and/or theorganic acid producing microorganism are recovered. The integratedprocesses according to the invention comprise recovery of all kinds ofproducts made during the integrated processes including fermentationproducts such as ethanol and succinic acid. A fermentation product maybe separated from the fermentation broth in manner know to the skilledperson. Examples of techniques for recovery include, but are not limitedto, chromatography, electrophoretic procedures, differential solubility,distillation, or extraction. For each fermentation product the skilledperson will thus be able to select a proper separation technique. Forinstance, ethanol may be separated from a yeast fermentation broth bydistillation, for instance steam distillation/vacuum distillation inconventional way.

In an embodiment the integrated processes of the invention also produceenergy, heat, electricity and/or steam.

In an embodiment the solid fraction obtained after solid/liquidseparation of the enzymatically hydrolysed lignocellulosic material, thewaste obtained after purification/recovery of the organic acid, and/orthe solids obtained after distillation/recovery of the ethanol can beused in the production of electricity. Electricity can be made byincineration of any one of the above-mentioned materials. Theelectricity can be used in any one of the steps of the integratedprocesses according to the present invention.

The beneficial effects of the present invention are found for severallignocellulosic materials and therefore believed to be present for thehydrolysis of all kind of lignocellulosic materials. This beneficialeffects of the present invention are found for several enzymes andtherefore believed to be present for all kind of hydrolysing enzymecompositions.

EXAMPLES Example 1

Integrated Process for Alcohol Production and Organic Acid Productionfrom Lignocellulosic Material

A single batch of pretreated lignocellulosic material was separated bycentrifugation into a solid fraction and liquid fraction. The solidfraction obtained after solid/liquid separation of the pretreatedlignocellulosic material was washed to obtain a cellulose-rich pulp.

Part of the pulp was subjected to enzymatic hydrolysis. In this case, 64kg dry matter pulp was hydrolysed in a 400 liter stirred vessel byadding it to 254 liter an aqueous composition containing cellulolyticenzymes from Rasamsonia (which was at a temperature of 62° C.). Thefirst dosage of pulp resulted in 10% w/w dry matter of pH 4.2, which wasliquefied by the enzymes within 3 hours. From that moment on, portionsof 5 kg dry matter pulp were added each hour until 350 kg mash wasobtained, while pH was adjusted to 4.2 with a 10% ammonia solution. Thehydrolysis was continued while stirring at 62° C. for another 4 days andresulted in a glucose-rich hydrolysate.

The hydrolysate was centrifuged to obtain a solid fraction and a liquidfraction. The solid fraction was washed with water. The wash water wasadded to the liquid fraction and the combined liquid fractions wereconcentrated by evaporation until a final concentrated liquid fractionwas obtained that contained glucose at a concentration of approximately450 g/kg.

Part of the concentrated liquid fraction was used for propagation ofgenetically modified succinic acid overproducing yeast of the genusSaccharomyces cerevisae. The medium for propagation of the yeast wasbased on Verduyn glucose medium and contained ammonium sulphate,potassium phosphate, magnesium phosphate, trace elements and vitaminsand 8 g/kg of the concentrated liquid fraction as carbon source (forVerduyn medium see Yeast 8, (1992), pages 201-517). Propagation was donefor 68 hours in a stirred vessel at 30° C. with continuous stirring.

The so-obtained seed culture was added to inoculate a fermentorcontaining Verduyn medium with, among other components such as urea,biotin and calcium carbonate in defined concentrations. As carbonsource, the concentrated liquid fraction was added by feeding it duringthe duration of the fermentation at a rate of 16 mL/kg·h. After 48hours, the fermentation was stopped and the broth was centrifuged. Thesupernatant was subjected to repeatedly evaporation, crystallization,polishing and drying, resulting in crude succinic acid crystals.

Another part of the concentrated liquid fraction was used forpropagation of an enzyme producing microorganism and production ofenzymes by the enzyme producing microorganism. A fermentor containingmineral medium with 20 g/kg concentrate and 40 g/kg solid dry matterpulp was inoculated with the fungus Rasamsonia emersonii. During thefirst phase of the fermentation process, also called the growth phase orpropagation phase, fungal biomass increases without protein production.In the second phase of the fermentation process, also called the enzymeproduction phase, enzymes are produced. The fermentation was performedunder aseptic aerobic conditions at 37° C. pH 6 for 120 hours, while theconcentrated liquid fraction was added as feed. The final proteinconcentration obtained at the end of fermentation was 65 g/kgsupernatant. The obtained supernatant showed cellulolytic activity.

A part of the liquid fraction obtained after solid/liquid separation ofthe pretreated lignocellulosic material was mixed with a part of theconcentrated liquid fraction to get a fermentable mixture. This mixturewas fermented with the pentose fermenting Saccharomyces cerevisae strainRN1016 and yielded 5.1% w/w ethanol after fermentation for 48 hours atpH 5.5.

In a separate experiment, glucose-rich hydrolysate as such was fermentedwith Saccharomyces cerevisae strain RN1016 in a 34 hour fermentation atpH 4.2. The yield of ethanol on sugars was 90%.

Example 2

Integrated Process for Alcohol Production from Lignocellulosic Material

A single batch of pretreated lignocellulosic material was separated bycentrifugation into a solid fraction and a liquid fraction. The liquidfraction was stored at 4° C. until use in the production of ethanol (seebelow). The solid fraction obtained after solid/liquid separation of thepretreated lignocellulosic material was washed to obtain acellulose-rich pulp.

Part of the pulp was subjected to enzymatic hydrolysis. In this case, 64kg dry matter pulp was hydrolysed in a 400 liter stirred vessel byadding it to 254 liter an aqueous composition containing cellulolyticenzymes from Rasamsonia (which was at a temperature of 62° C.). Thefirst dosage of pulp resulted in 10% w/w dry matter of pH 4.2, which wasliquefied by the enzymes within 3 hours. From that moment on, portionsof 5 kg dry matter pulp were added each hour until 350 kg mash wasobtained, while pH was adjusted to 4.2 with a 10% ammonia solution. Thehydrolysis was continued while stirring at 62° C. for another 4 days andresulted in a glucose-rich hydrolysate.

The glucose-rich hydrolysate was centrifuged to obtain a solid fractionand a liquid fraction. The solid fraction was washed with water. Thewash water was added to the liquid fraction and the combined liquidfractions were concentrated by evaporation until a final concentratedliquid fraction was obtained that contained glucose at a concentrationof approximately 450 g/kg.

The liquid fraction obtained after pretreatment of the lignocellulosicmaterial was divided into four equal portions. The first portion waskept as it is (undiluted portion) and fermented as such. The secondportion was diluted to 70% w/w of its original concentration with waterand fermented. The third portion was diluted to 70% w/w with 13% w/wconcentrated liquid fraction and 17% w/w water and fermented. The fourthportion was diluted to 70% w/w with 20% w/w concentrated liquid fractionand 10% w/w water and fermented.

The portions were fermented with the pentose fermenting Saccharomycescerevisae strain RN1016 for 48 hours at pH 5.5 to produce ethanol andthe ethanol concentration was measured using HPLC. The measured ethanolconcentration is expressed as % w/w fermented material at the end of thefermentation. The results are shown in Table 1.

Table 1 shows that diluting the liquid fraction obtained afterpretreatment of the lignocellulosic material results in a higher ethanolyield. Table 1 also shows that even higher ethanol production yieldswere obtained when the liquid fraction obtained after pretreatment ofthe lignocellulosic material was diluted with a concentrated liquidfraction obtained after solid/liquid separation of the hydrolysatecompared to when the liquid fraction obtained after pretreatment of thelignocellulosic material was diluted with water only.

Example 3

Integrated Process for Alcohol Production from Lignocellulosic Material

The example is done essentially as described in Example 2 with theproviso that the dilution is not done with the concentrated liquidfraction, but with the solid fraction obtained after solid/liquidseparation of the hydrolysate. The solid fraction contains residualsoluble sugars (about 17% w/w of total solids in the solid fraction)that are entrapped in the remaining insoluble sugar fraction and ligninpresent in the solid fraction.

The liquid fraction is diluted to 70% w/w/with the solid fraction.Dilution with the solid fraction results in a higher ethanolconcentration (about 4% w/w ethanol).

Example 4 Integrated Process for Production of Enzymes by an EnzymeProducing Microorganism

A single batch of pretreated lignocellulosic material was separated bycentrifugation into a solid fraction and a liquid fraction. The solidfraction obtained after solid/liquid separation of the pretreatedlignocellulosic material was washed to obtain cellulose-rich solids.

Part of the solids was used as a substrate (called substrate A) toinduce enzyme production. Another part of the solids was subjected toenzymatic hydrolysis as described in Example 1. The hydrolysate obtainedafter enzymatic hydrolysis was centrifuged to obtain a solid fractionand a liquid fraction. The solid fraction (called substrate B) waswashed with water and used to induce enzyme production.

The wash water was added to the liquid fraction and the resulting liquidfraction was concentrated by evaporation until a final concentratedliquid fraction was obtained that contained glucose at a concentrationof approximately 450 g/kg.

The concentrated liquid fraction was used as a carbon source in twoenzyme production processes in the fungus Rasamsonia. In one process,substrate A was used as enzyme production inducer, while in the otherprocess, substrate B was used as an enzyme production inducer. Theproduction processes consisted of a growth phase and an enzymeproduction phase. At the end of the enzyme production phase, the amountof enzyme present in the liquid fraction of the fermentation broth wasdetermined using a standard protein determination assay and showed to beof a comparable level (50+/−5 g/L), demonstrating that both the solidfraction obtained after solid/liquid separation of pretreatedlignocellulosic material as such and the solid fraction obtained aftersolid/liquid separation of an enzymatic hydrolysate can be used asinducer in enzyme production.

TABLE 1 Ethanol production after a fermentation of 48 hours of dilutedand undiluted portions. Ethanol concentration (in % w/w) Portion 1(undiluted) 1.2 Portion 2 (diluted to 70% with water) 2.0 Portion 3(diluted to 70% with 13% 4.5 concentrated liquid fraction and 17% water)Portion 4 (diluted to 70% with 20% 5.3 concentrated liquid fraction and10% water)

1. An integrated process for alcohol production from lignocellulosicmaterial, wherein the process comprises: enzymatic hydrolysis of thelignocellulosic material to obtain enzymatically hydrolysedlignocellulosic material, solid/liquid separation of the enzymaticallyhydrolysed lignocellulosic material to obtain at least a solid fractionand at least a liquid fraction, fermentation of the solid fractionand/or the liquid fraction by an alcohol producing microorganism toproduce alcohol, propagation of the alcohol producing microorganism byfermentation of the liquid fraction and/or the solid fraction,propagation of an enzyme producing microorganism, and production ofenzymes by the enzyme producing microorganism.
 2. Process according toclaim 1, wherein a part of the enzymatically hydrolysed lignocellulosicmaterial and a part of the lignocellulosic material is used in thepropagation of the enzyme producing microorganism and/or the productionof enzymes by the enzyme producing microorganism.
 3. Process accordingto claim 1, wherein the enzymes produced by the enzyme producingmicroorganism are used in the enzymatic hydrolysis.
 4. Process accordingto claim 1, wherein the at least solid fraction comprises between 3 and97 wt % C5 sugars.
 5. Process according to claim 1, wherein the at leastliquid fraction comprises between 1 and 97 wt % C6 sugars.
 6. Processaccording to claim 1, wherein the enzymatic hydrolysis comprises atleast: a liquefaction step wherein the lignocellulosic material ishydrolysed in at least a first container, and a saccharification stepwherein the liquefied lignocellulosic material is hydrolysed in the atleast first container and/or in at least a second container.
 7. Processaccording to claim 1, wherein the alcohol, the enzymes, the enzymeproducing microorganism and/or the alcohol producing microorganism arerecovered.
 8. Process according to claim 1, wherein the lignocellulosicmaterial is subjected to at least one solid/liquid separation before theenzymatic hydrolysis.
 9. Process according to claim 1, wherein oxygen isadded during the enzymatic hydrolysis.
 10. Process according to claim 1,wherein the enzyme producing microorganism is a fungus.
 11. Processaccording to claim 10, wherein the fungus is Rasamsonia.
 12. Processaccording to claim 1, wherein the enzymes are in the form of a wholefermentation broth.
 13. Process according to claim 1, wherein thealcohol producing microorganism is able to ferment at least a C5 sugarand at least a C6 sugar.
 14. Process according to claim 13, wherein thealcohol producing microorganism is a yeast.
 15. Process according toclaim 13, wherein the alcohol producing microorganism is Saccharomycescerevisiae.
 16. Process according to claim 1, wherein the dry mattercontent at the end of the enzymatic hydrolysis is 5 wt % or higher. 17.Process according to claim 1, wherein the alcohol is ethanol.